Modulation of mitochondrial ribosomal protein L13 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of mitochondrial ribosomal protein L13. The compositions comprise oligonucleotides, targeted to nucleic acid encoding mitochondrial ribosomal protein L13. Methods of using these compounds for modulation of mitochondrial ribosomal protein L13 expression and for diagnosis and treatment of disease associated with expression of mitochondrial ribosomal protein L13 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of mitochondrial ribosomal protein L13. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding mitochondrial ribosomal protein L13. Such compounds are shown herein to modulate the expression of mitochondrial ribosomal protein L13.

BACKGROUND OF THE INVENTION

[0002] The mitochondria is an organelle containing its own highly conserved genome which codes for 13 polypeptides essential for oxidative phosphorylation. The mitochondria is therefore responsible for the synthesis of the majority of the ATP used by eukaryotic organisms. The mitochondria have their own ribosomes, called mitoribosomes, to translate the genes encoded by the mitochondrial DNA. The mitoribosome is composed of two RNA species, also encoded by the mitochondrial genome, and over 70 proteins, encoded by nuclear DNA, and these mitochondrial ribosome proteins (MRPs) are assembled into a large and small subunit. A wide variety of diseases such as diabetes, optic neuropathy, deafness, neuromuscular disorders and cancer have been linked to defects in mitochondrial processes. Such diseases also result from defects in proteins targeted to the mitochondria or mutations in the mitochondrial DNA, which occur at a higher rate than mutations in nuclear DNA (Wallace, Science, 1999, 283, 1482-1488). In addition, the MRPs may have additional, potentially pathological roles in the cell, as demonstrated by MRP-S29 (also known as death-associated protein 3) which is involved in apoptosis (Kissil et al., EMBO J., 1999, 18, 353-362).

[0003] Recently many of the genes encoding mammalian mitochondrial ribosomal proteins have been cloned, including the human gene for one member of the large subunit named mitochondrial ribosome protein L13 (Suzuki et al., J. Biol. Chem., 2001, 276, 21724-21736). Mitochondrial ribosome protein L13 (also called L13 protein, and MRPL13) shares 97% homology to the analogous mouse protein and a smaller percent homology (35 to 48%) to the analogous proteins from C. elegans, S. cerevisiae, E. coli, B. subtilis, and M. pneumoniae (Suzuki et al., J. Biol. Chem., 2001, 276, 21724-21736). The gene has been localized to chromosome 8q22.1-q22.3 (Kenmochi et al., Genomics, 2001, 77, 65-70).

[0004] Defects in the mitochondrial translational apparatus can result in many abnormal phenotypes, thus defects in the MRPs have been considered candidates for a variety of pathological conditions. Currently, there are no known therapeutic agents which effectively inhibit the synthesis of mitochondrial ribosome protein L13 and to date, no investigative strategies aimed at modulating mitochondrial ribosome protein L13 function have been reported. Consequently, there remains a long felt need for agents capable of effectively inhibiting mitochondrial ribosome protein L13 function.

[0005] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of mitochondrial ribosome protein L13 expression.

[0006] The present invention provides compositions and methods for modulating mitochondrial ribosome protein L13 expression.

SUMMARY OF THE INVENTION

[0007] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding mitochondrial ribosomal protein L13, and which modulate the expression of mitochondrial ribosomal protein L13. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of mitochondrial ribosomal protein L13 and methods of modulating the expression of mitochondrial ribosomal protein L13 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of mitochondrial ribosomal protein L13 are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0008] A. Overview of the Invention

[0009] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding mitochondrial ribosomal protein L13. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding mitochondrial ribosomal protein L13. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding mitochondrial ribosomal protein L13” have been used for convenience to encompass DNA encoding mitochondrial ribosomal protein L13, RNA (including pre-mRNA and MRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0010] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of mitochondrial ribosomal protein L13. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and MRNA is often a preferred target nucleic acid.

[0011] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0012] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0013] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0014] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0015] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0016] B. Compounds of the Invention

[0017] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0018] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0019] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0020] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0021] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0022] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0023] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0024] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0025] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0026] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0027] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0028] C. Targets of the Invention

[0029] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes mitochondrial ribosomal protein L13.

[0030] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0031] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed MRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding mitochondrial ribosomal protein L13, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0032] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0033] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0034] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an MRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0035] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0036] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0037] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “MRNA variants”. Consequently, MRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0038] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or MRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0039] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0040] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0041] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0042] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0043] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0044] D. Screening and Target Validation

[0045] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of mitochondrial ribosomal protein L13. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding mitochondrial ribosomal protein L13 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred-target segment of a nucleic acid molecule -encoding mitochondrial ribosomal protein L13 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding mitochondrial ribosomal protein L13. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding mitochondrial ribosomal protein L13, the modulator may then be employed in further investigative studies of the function of mitochondrial ribosomal protein L13, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0046] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0047] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0048] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between mitochondrial ribosomal protein L13 and a disease state, phenotype, or condition. These methods include detecting or modulating mitochondrial ribosomal protein L13 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of mitochondrial ribosomal protein L13 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0049] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0050] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0051] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0052] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0053] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0054] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding mitochondrial ribosomal protein L13. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective mitochondrial ribosomal protein L13 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding mitochondrial ribosomal protein L13 and in the amplification of said nucleic acid molecules for detection or for use in further studies of mitochondrial ribosomal protein L13. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding mitochondrial ribosomal protein L13 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of mitochondrial ribosomal protein L13 in a sample may also be prepared.

[0055] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0056] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of mitochondrial ribosomal protein L13 is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a mitochondrial ribosomal protein L13 inhibitor. The mitochondrial ribosomal protein L13 inhibitors of the present invention effectively inhibit the activity of the mitochondrial ribosomal protein L13 protein or inhibit the expression of the mitochondrial ribosomal protein L13 protein. In one embodiment, the activity or expression of mitochondrial ribosomal protein L13 in an animal is inhibited by about 10%. Preferably, the activity or expression of mitochondrial ribosomal protein L13 in an animal is inhibited by about 30%. More preferably, the activity or expression of mitochondrial ribosomal protein L13 in an animal is inhibited by 50% or more.

[0057] For example, the reduction of the expression of mitochondrial ribosomal protein L13 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed contain a nucleic acid molecule encoding mitochondrial ribosomal protein L13 protein and/or the mitochondrial ribosomal protein L13 protein itself.

[0058] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0059] F. Modifications

[0060] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0061] Modified Internucleoside Linkages (Backbones)

[0062] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0063] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0064] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0065] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0066] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0067] Modified Sugar and Internucleoside Linkages-Mimetics

[0068] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0069] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0070] Modified Sugars

[0071] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0072] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F) The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0073] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0074] Natural and Modified Nucleobases

[0075] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡—C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0076] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0077] Conjugates

[0078] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/U.S.92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0079] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0080] Chimeric Compounds

[0081] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0082] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0083] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0084] G. Formulations

[0085] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0086] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0087] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0088] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0089] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0090] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0091] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0092] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0093] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0094] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0095] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0096] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0097] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0098] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0099] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0100] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0101] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. applications Ser. Nos. 09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0102] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0103] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0104] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0105] H. Dosing

[0106] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0107] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0108] Synthesis of Nucleoside Phosphoramidites

[0109] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylamino-oxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine , 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nuclebside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0110] Oligonucleotide and Oligonucleoside Synthesis

[0111] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0112] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0113] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0114] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0115] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0116] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0117] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/U.S.94/00902 and PCT/U.S.93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0118] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0119] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0120] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0121] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0122] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0123] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0124] RNA Synthesis

[0125] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0126] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0127] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0128] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1, 1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′- groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0129] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethyl-hydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0130] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0131] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0132] Synthesis of Chimeric Oligonucleotides

[0133] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0134] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0135] [2′-O-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[2′-O-(2-Methoxyethyl) Phosphodiester]--[2′-deoxy Phosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides

[0136] [2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxy phosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0137] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0138] Design and Screening of Duplexed Antisense Compounds Targeting Mitochondrial Ribosomal Protein L13

[0139] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target mitochondrial ribosomal protein L13. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0140] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine (dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   | | | | | | | | | | | | | | | | | | | TTgctctccgcctgccctggc Complement

[0141] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0142] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate mitochondrial ribosomal protein L13 expression.

[0143] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0144] Oligonucleotide Isolation

[0145] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0146] Oligonucleotide Synthesis—96 Well Plate Format

[0147] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0148] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0149] Oligonucleotide Analysis—96-Well Plate Format

[0150] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0151] Cell Culture and Oligonucleotide Treatment

[0152] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0153] T-24 Cells:

[0154] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0155] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0156] A549 Cells:

[0157] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0158] NHDF Cells:

[0159] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0160] HEK Cells:

[0161] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0162] Treatment with Antisense Compounds:

[0163] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™0 (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0164] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0165] Analysis of Oligonucleotide Inhibition of Mitochondrial Ribosomal Protein L13 Expression

[0166] Antisense modulation of mitochondrial ribosomal protein L13 expression can be assayed in a variety of ways known in the art. For example, mitochondrial ribosomal protein L13 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ MRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0167] Protein levels of mitochondrial ribosomal protein L13 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to mitochondrial ribosomal protein L13 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0168] Design of Phenotypic Assays and In Vivo Studies for the use of Mitochondrial Ribosomal Protein L13 Inhibitors

[0169] Phenotypic Assays

[0170] Once mitochondrial ribosomal protein L13 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of mitochondrial ribosomal protein L13 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0171] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with mitochondrial ribosomal protein L13 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0172] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0173] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the mitochondrial ribosomal protein L13 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0174] In Vivo Studies

[0175] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0176] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or mitochondrial ribosomal protein L13 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a mitochondrial ribosomal protein L13 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0177] Volunteers receive either the mitochondrial ribosomal protein L13 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding mitochondrial ribosomal protein L13 or mitochondrial ribosomal protein L13 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0178] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0179] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and mitochondrial ribosomal protein L13 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the mitochondrial ribosomal protein L13 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0180] RNA Isolation

[0181] Poly(A)+ mRNA isolation

[0182] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ MRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0183] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0184] Total RNA Isolation

[0185] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0186] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0187] Real-time Quantitative PCR Analysis of Mitochondrial Ribosomal Protein L13 mRNA Levels

[0188] Quantitation of mitochondrial ribosomal protein L13 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0189] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target MRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0190] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5× PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5× ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0191] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0192] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0193] Probes and primers to human mitochondrial ribosomal protein L13 were designed to hybridize to a human mitochondrial ribosomal protein L13 sequence, using published sequence information (GenBank accession number NM_(—)014078.1, incorporated herein as SEQ ID NO:4). For human mitochondrial ribosomal protein L13 the PCR primers were: forward primer: AAGGACGTACGGTCCTGCTAGTA (SEQ ID NO: 5) reverse primer: TCTAGCAAAAGTGGCCCATTG (SEQ ID NO: 6) and the PCR probe was: FAM-AGGAATATGTCGAGTTTCTCTAGGGCGCC-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0194] Northern Blot Analysis of Mitochondrial Ribosomal Protein L13 mRNA Levels

[0195] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0196] To detect human mitochondrial ribosomal protein L13, a human mitochondrial ribosomal protein L13 specific probe was prepared by PCR using the forward primer AAGGACGTACGGTCCTGCTAGTA (SEQ ID NO: 5) and the reverse primer TCTAGCAAAAGTGGCCCATTG (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0197] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0198] Antisense Inhibition of Human Mitochondrial Ribosomal Protein L13 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0199] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human mitochondrial ribosomal protein L13 RNA, using published sequences (GenBank accession number NM_(—)014078.1, incorporated herein as SEQ ID NO: 4, and the complement of nucleotides 54000 to 106000 of the sequence with GenBank accession number NT_(—)031821.2, incorporated herein as SEQ ID NO: 11). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-thyl methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are methylcytidines. The compounds were analyzed for their effect on human mitochondrial ribosomal protein L13 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which T-24 cells were treated with the antisense olignucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human mitochondrial ribosomal protein L13 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB ID NO NO 282274 Start 4 36 agaaactcgacatattcctc 81 12 1 Codon 282275 Start 4 37 gagaaactcgacatattcct 70 13 1 Codon 282276 Coding 4 70 ctagcaaaagtggcccattg 95 14 1 282277 Coding 4 76 catattctagcaaaagtggc 61 15 1 282278 Coding 4 95 tttcccatctaagagatacc 75 16 1 282279 Coding 4 96 ttttcccatctaagagatac 63 17 1 282280 Coding 4 106 ggtggctgcattttcccatc 94 18 1 282281 Coding 4 107 aggtggctgcattttcccat 92 19 1 282282 Coding 4 111 tgccaggtggctgcattttc 94 20 1 282283 Coding 4 115 agtttgccaggtggctgcat 92 21 1 282284 Coding 4 119 agcaagtttgccaggtggct 93 22 1 282285 Coding 4 128 tgccatagcagcaagtttgc 90 23 1 282286 Coding 4 129 atgccatagcagcaagtttg 93 24 1 282287 Coding 4 139 agtcttatagatgccatagc 93 25 1 282288 Coding 4 157 ggtttatgtaatccctgaag 92 26 1 282289 Coding 4 158 aggtttatgtaatccctgaa 86 27 1 282290 Coding 4 163 tacacaggtttatgtaatcc 61 28 1 282291 Coding 4 165 ggtacacaggtttatgtaat 84 29 1 282292 Coding 4 167 atggtacacaggtttatgta 84 30 1 282293 Coding 4 170 tgcatggtacacaggtttat 92 31 1 282295 Coding 4 172 agtgcatggtacacaggttt 93 32 1 282297 Coding 4 176 actcagtgcatggtacacag 92 33 1 282299 Coding 4 184 ccacagtcactcagtgcatg 90 34 1 282301 Coding 4 198 taacaacatgatccccaacag 80 35 1 282304 Coding 4 209 tgtgttcattataacaacat 87 36 1 282306 Coding 4 213 gtcttgtgttcattataaca 90 37 1 282308 Coding 4 222 atgcaatgtgtcttgtgttc 90 38 1 282310 Coding 4 225 aaaatgcaatgtgtcttgtg 89 39 1 282312 Coding 4 261 gcgaagagtatactttttgt 84 40 1 282314 Coding 4 265 gtatgcgaagagtatacttt 78 41 1 282316 Coding 4 266 agtatgcgaagagtatactt 75 42 1 282318 Coding 4 267 cagtatgcgaagagtatact 79 43 1 282320 Coding 4 274 gggtagccagtatgcgaaga 71 44 1 282322 Coding 4 278 acctgggtagccagtatgcg 83 45 1 282324 Coding 4 304 tgagcagctgttacttgtct 90 46 1 282326 Coding 4 305 ctgagcagctgttacttgtc 44 47 1 282328 Coding 4 326 cactggatccctcaggtgaa 82 48 1 282330 Coding 4 330 ttgccactggatccctcagg 83 49 1 282332 Coding 4 339 gttttacaattgccactgga 81 50 1 282334 Coding 4 353 gccataaatagctagtttta 86 51 1 282336 Coding 4 362 tggcagcatgccataaatag 72 52 1 282338 Coding 4 366 tttttggcagcatgccataa 72 53 1 282340 Coding 4 387 tcattgttcttctgtgaagg 65 54 1 282342 Coding 4 390 ccatcattgttcttctgtga 77 55 1 282344 Coding 4 400 tgcaacctttccatcattgt 87 56 1 282346 Coding 4 435 gaatatcttctggaatatac 58 57 1 282348 Coding 4 448 actaaattcttaagaatatc 14 58 1 282350 Coding 4 452 ctctactaaattcttaagaa 3 59 1 282352 Coding 4 470 tcgtggttgaggaagctcct 83 60 1 282354 Coding 4 479 aggtatttttcgtggttgag 87 61 1 282356 Coding 4 501 ttgtgtactcatctagacgt 81 62 1 282358 Coding 4 504 ttcttgtgtactcatctaga 72 63 1 282360 Coding 4 536 ggagtccacaatcttggaaa 74 64 1 282362 Coding 4 538 gtggagtccacaatcttgga 76 65 1 282364 Stop 4 542 tcaggtggagtccacaatct 62 66 1 Codon 282366 Stop 4 545 tcttcaggtggagtccacaa 58 67 1 Codon 282368 Stop 4 550 gatattcttcaggtggagtc 47 68 1 Codon 282370 Stop 4 557 tatagccgatattcttcagg 52 69 1 Codon 282372 3′UTR 4 562 tctcttatagccgatattct 67 70 1 282374 3′UTR 4 597 ttcaatcacttcactgttat 90 71 1 282376 3′UTR 4 612 tcatcagaagaaagtttcaa 53 72 1 282378 3′UTR 4 625 acgttaaagaaactcatcag 17 73 1 282380 3′UTR 4 659 gctgaactgtaccatttgtt 39 74 1 282382 3′UTR 4 661 gtgctgaactgtaccatttg 37 75 1 282384 3′UTR 4 664 caggtgctgaactgtaccat 72 76 1 282386 3′UTR 4 666 aacaggtgctgaactgtacc 80 77 1 282388 3′UTR 4 673 cacataaaacaggtgctgaa 77 78 1 282390 3′UTR 4 694 cctttccccacagtgattca 82 79 1 282392 3′UTR 4 712 aggactacacctttgacc 77 80 1 282394 3′UTR 4 714 gaaggactacaccttcctga 90 81 1 282396 3′UTR 4 766 gattacataaaaatggttct 70 82 1 282398 exon: 11 14467 ttacccagtatgcgaagagt 84 83 1 intron junction 282400 exon: 11 14473 attatcttacccagtatgcg 67 84 1 intron junction 282402 exon 11 21194 aacttactgccactggatcc 54 85 1 282404 exon 11 21197 agaaacttactgccactgga 53 86 1 282406 exon: 11 26642 cctcatctggaaaaagatgc 48 87 1 intron junction 282408 inton 11 37608 aagaggatgctcaccaatgc 85 88 1 282410 intron 11 43906 tgggcagatttcatgacaaa 72 89 1

[0200] As shown in Table 1, SEQ ID NOs 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 60, 61, 62, 63, 64, 65, 66, 67, 69, 70, 71, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88 and 89 demonstrated at least 50% inhibition of human mitochondrial ribosomal protein L13 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs 18, 32 and 81. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in mitochondrial ribosomal protein L13. TARGET SITE SEQ ID TARGET REV COMP SEQ ID ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 198412 4 36 gaggaatatgtcgagtttct 12 H. sapiens 90 198413 4 37 aggaatatgtcgagtttctc 13 H. sapiens 91 198414 4 70 caatgggccacttttgctag 14 H. sapiens 92 198415 4 76 gccacttttgctagaatatg 15 H. sapiens 93 198416 4 95 ggtatctcttagatgggaaa 16 H. sapiens 94 198417 4 96 gtatctcttagatgggaaaa 17 H. sapiens 95 198418 4 106 gatgggaaaatgcagccacc 18 H. sapiens 96 198419 4 107 atgggaaaatgcagccacct 19 H. sapiens 97 198420 4 111 gaaaatgcagccacctggca 20 H. sapiens 98 198421 4 115 atgcagccacctggcaaact 21 H. sapiens 99 198422 4 119 agccacctggcaaacttgct 22 H. sapiens 100 198423 4 128 gcaaacttgctgctatggca 23 H. sapiens 101 198424 4 129 caaacttgctgctatggcat 24 H. sapiens 102 198425 4 139 gctatggcatctataagact 25 H. sapiens 103 198426 4 157 cttcagggattacataaacc 26 H. sapiens 104 198427 4 158 ttcagggattacataaacct 27 H. sapiens 105 198428 4 163 ggattacataaacctgtgta 28 H. sapiens 106 198429 4 165 attacataaacctgtgtacc 29 H. sapiens 107 198430 4 167 tacataaacctgtgtaccat 30 H. sapiens 108 198431 4 170 ataaacctgtgtaccatgca 31 H. sapiens 109 198432 4 172 aaacctgtgtaccatgcact 32 H. sapiens 110 198433 4 176 ctgtgtaccatgcactgagt 33 H. sapiens 111 198434 4 184 catgcactgagtgactgtgg 34 H. sapiens 112 198435 4 198 ctgtggggatcatgttgtta 35 H. sapiens 113 198436 4 209 atgttgttataatgaacaca 36 H. sapiens 114 198437 4 213 tgttataatgaacacaagac 37 H. sapiens 115 198438 4 222 gaacacaagacacattgcat 38 H. sapiens 116 198439 4 225 cacaagacacattgcatttt 39 H. sapiens 117 198440 4 261 acaaaaagtatactcttcgc 40 H. sapiens 118 198441 4 265 aaagtatactcttcgcatac 41 H. sapiens 119 198442 4 266 aagtatactcttcgcatact 42 H. sapiens 120 198443 4 267 agtatactcttcgcatactg 43 H. sapiens 121 198444 4 274 tcttcgcatactggctaccc 44 H. sapiens 122 198445 4 278 cgcatactggctacccaggt 45 H. sapiens 123 198446 4 304 aqacaagtaacagctgctca 46 H. sapiens 124 198448 4 326 ttcacctgagggatccagtg 48 H. sapiens 125 198449 4 330 cctgagggatccagtggcaa 49 H. sapiens 126 198450 4 339 tccagtggcaattgtaaaac 50 H. sapiens 127 198451 4 353 taaaactagctatttatggc 51 H. sapiens 128 198452 4 362 ctatttatggcatgctgcca 52 H. sapiens 129 198453 4 366 ttatggcatgctgccaaaaa 53 H. sapiens 130 198454 4 387 ccttcacagaagaacaatga 54 H. sapiens 131 198455 4 390 tcacagaagaacaatgatgg 55 H. sapiens 132 198456 4 400 acaatgatggaaaggttgca 56 H. sapiens 133 198457 4 435 gtatattccagaagatattc 57 H. sapiens 134 198460 4 470 aggagcttcctcaaccacga 60 H. sapiens 135 198461 4 479 ctcaaccacgaaaaatacct 61 H. sapiens 136 198462 4 501 acgtctagatgagtacacaa 62 H. sapiens 137 198463 4 504 tctagatgagtacacaagaa 63 H. sapiens 138 198464 4 536 tttccaagattgtggactcc 64 H. sapiens 139 198465 4 538 tccaagattgtggacticcac 65 H. sapiens 140 198466 4 542 agattgtggactccacctga 66 H. sapiens 141 198467 4 545 ttgtggactccacctgaaga 67 H. sapiens 142 198469 4 557 cctgaagaatatcggctata 69 H. sapiens 143 198470 4 562 agaatatcggctataagaga 70 H. sapiens 144 198471 4 597 ataacagtgaagtgattgaa 71 H. sapiens 145 198472 4 612 ttgaaactttcttctgatga 72 H. sapiens 146 198476 4 664 atggtacagttcagcacctg 76 H. sapiens 147 198477 4 666 ggtacagttcagcacctgtt 77 H. sapiens 148 198478 4 673 ttcagcacctgttttatgtg 78 H. sapiens 149 198479 4 694 tgaatcactgtggggaaagg 79 H. sapiens 150 198480 4 712 ggtcaggaaggtgtagtcct 80 H. sapiens 151 198481 4 714 tcaggaaggtgtagtccttc 81 H. sapiens 152 198482 4 766 agaaccatttttatgtaatc 82 H. sapiens 153 198483 11 14467 actcttcgcatactgggtaa 83 H. sapiens 154 198484 11 14473 cgcatactgggtaagataat 84 H. sapiens 155 198485 11 21194 ggatccagtggcagtaagtt 85 H. sapiens 156 198486 11 21197 tccagtggcagtaagtttct 86 H. sapiens 157 198488 11 37608 gcattggtgagcatcctctt 88 H. sapiens 158 198489 11 43906 tttgtcatgaaatctgccca 89 H. sapiens 159

[0201] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of mitochondrial ribosomal protein L13.

[0202] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0203] Western Blot Analysis of Mitochondrial Ribosomal Protein L13 Protein Levels

[0204] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to mitochondrial ribosomal protein L13 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 159 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 862 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (43)...(561) <400> SEQUENCE: 4 ggctactagg agaaggacgt acggtcctgc tagtagagga at atg tcg agt ttc 54 Met Ser Ser Phe 1 tct agg gcg ccc cag caa tgg gcc act ttt gct aga ata tgg tat ctc 102 Ser Arg Ala Pro Gln Gln Trp Ala Thr Phe Ala Arg Ile Trp Tyr Leu 5 10 15 20 tta gat ggg aaa atg cag cca cct ggc aaa ctt gct gct atg gca tct 150 Leu Asp Gly Lys Met Gln Pro Pro Gly Lys Leu Ala Ala Met Ala Ser 25 30 35 ata aga ctt cag gga tta cat aaa cct gtg tac cat gca ctg agt gac 198 Ile Arg Leu Gln Gly Leu His Lys Pro Val Tyr His Ala Leu Ser Asp 40 45 50 tgt ggg gat cat gtt gtt ata atg aac aca aga cac att gca ttt tct 246 Cys Gly Asp His Val Val Ile Met Asn Thr Arg His Ile Ala Phe Ser 55 60 65 gga aac aaa tgg gaa caa aaa gta tac tct tcg cat act ggc tac cca 294 Gly Asn Lys Trp Glu Gln Lys Val Tyr Ser Ser His Thr Gly Tyr Pro 70 75 80 ggt gga ttt aga caa gta aca gct gct cag ctt cac ctg agg gat cca 342 Gly Gly Phe Arg Gln Val Thr Ala Ala Gln Leu His Leu Arg Asp Pro 85 90 95 100 gtg gca att gta aaa cta gct att tat ggc atg ctg cca aaa aac ctt 390 Val Ala Ile Val Lys Leu Ala Ile Tyr Gly Met Leu Pro Lys Asn Leu 105 110 115 cac aga aga aca atg atg gaa agg ttg cat ctt ttt cca gat gag tat 438 His Arg Arg Thr Met Met Glu Arg Leu His Leu Phe Pro Asp Glu Tyr 120 125 130 att cca gaa gat att ctt aag aat tta gta gag gag ctt cct caa cca 486 Ile Pro Glu Asp Ile Leu Lys Asn Leu Val Glu Glu Leu Pro Gln Pro 135 140 145 cga aaa ata cct aaa cgt cta gat gag tac aca aga aga aat aga cgc 534 Arg Lys Ile Pro Lys Arg Leu Asp Glu Tyr Thr Arg Arg Asn Arg Arg 150 155 160 ctt tcc aag att gtg gac tcc acc tga agaatatcgg ctataagaga 581 Leu Ser Lys Ile Val Asp Ser Thr 165 170 ataagaatgc agaaaataac agtgaagtga ttgaaacttt cttctgatga gtttctttaa 641 cgtacaggat ggagtaaaac aaatggtaca gttcagcacc tgttttatgt gttgaatcac 701 tgtggggaaa ggtcaggaag gtgtagtcct tcaataggaa attgtaatta aaatataatt 761 ttatagaacc atttttatgt aatctgattt gaatgttata gttgataata ataaaatcac 821 ttacttggtt gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 862 <210> SEQ ID NO 5 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 aaggacgtac ggtcctgcta gta 23 <210> SEQ ID NO 6 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 tctagcaaaa gtggcccatt g 21 <210> SEQ ID NO 7 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 aggaatatgt cgagtttctc tagggcgcc 29 <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH: 52001 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11121, 11122, 11123, 11124, 11125, 11126, 11127, 11128, 11129, 11130, 11131, 11132, 11133, 11134, 11135, 11136, 11137, 11138, 11139, 11140, 11141, 11142, 11143, 11144, 11145, 11146, 11147, 11148, 11149, 11150, 11151, 11152, 11153 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11154, 11155, 11156, 11157, 11158, 11159, 11160, 11161, 11162, 11163, 11164, 11165, 11166, 11167, 11168, 11169, 11170, 11171, 11172, 11173, 11174, 11175, 11176, 11177, 11178, 11179, 11180, 11181, 11182, 11183, 11184, 11185, 11186 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11187, 11188, 11189, 11190, 11191, 11192, 11193, 11194, 11195, 11196, 11197, 11198, 11199, 11200, 11201, 11202, 11203, 11204, 11205, 11206, 11207, 11208, 11209, 11210, 11211, 11212, 11213, 11214, 11215, 11216, 11217, 11218, 11219 <223> OTHER INFORMATION: n = A,T,C or G <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 11220 <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 11 tcatgataaa atcatcctca taaagccaca ctccccttag ttatacagaa ctaagatagg 60 caagaaaatt tcataaaagg agcaaaattc aagctgagaa cctagaataa ttttcataac 120 tttactatat tcaagcatct agaagaaagg ggtaatttat ttttcagttt tgtactccct 180 tagttatctc tgtaactcag tgccttgtac acagtcaaga agtatttcct tgtactcctt 240 agggtccaaa ttaattaagt taatttccag tacaaatgct gtgccagaga aaagaggagt 300 ttattatact taccagggaa agtactatct tctggattaa ttgaagcact aatgcttctt 360 ttcaagaggt gataaacatt tgctgctgtg aagtctgaaa aagaaaagat agactaactc 420 aaagggctct agatttacat aatctaactt agtccaattt caccatcaga gactctcaag 480 accagtaggt tgtacataaa agtactaaac gctgctcaag atctgttcaa gttttgatac 540 agaaaatcta aagcaatcag agaaaataga aaaggctccc gatctgtaca tgccagtcag 600 ttagcacgaa tgcaaaattc ccctcaaaag aataatttta aagagaaggg aaattctgtg 660 acaatacctt atacacaacc acatacattt actcctcaaa atgcctgttt gatcgaaaca 720 aatgaacaca taaaatgcaa tattgaatct agacgggtgc accaaaaagc agtgcaatcg 780 caaacaatta aaatcattta aaattcatca tttgttttta aaacttaggt tatatgtggg 840 atgtctcctc ccattagatg taaattgtag gacagcaaga tctttgtcca tcttgctcct 900 ttccgtatta tttgcatgtg aaacacagga gcagaaataa taaatgcata tatttccaaa 960 aagcaggaaa ttccattact aaggcataga aaatttatta agttaaacat ttactatgcc 1020 aagaacttct aagggctgga atcttgcatc cacaaatttg tccctgtttt tccatgaaga 1080 tgctattttt gcagccctca ttaactctat cccagataga gtcccagctt cgtgggacaa 1140 ctacatattg atccctcttc agcagaactt gatcacaaaa ctcaaacttc ttcccctctc 1200 cacaagccgt tcccgctctt tctcatccca gcgcttaccc ggctgattct cagtaccctc 1260 acccgacgac acctctggcc catgttctgc ctccctactg gccgccgacg ggaattttcc 1320 ttccccccag atcaccagca gcaggtaccg atccatctcc tcagagatca ccccccaact 1380 ttaggtctcg gcttccacat ccaaacagac gcgcgctatg acgcatttcc ggttcccttc 1440 gtttaggtcg gctggaaatt atgtcctccg tcggttttcc gcagtttttc caccaagcga 1500 gatatttttg ggagttattc cctaaataac tgcattatat gctcctttca tgacgaaatt 1560 gctgccgtgg agaagactgg aggaaactcg aggaagaggg agaagccgac aagtgctcga 1620 cgggctagga actgtcctgc ttgggtgtta gcgtttcccg ccgggccagt aaggctgagt 1680 gacccggcgt ggctactagg agaaggacgt acggtcctgc tagtagagga atatgtcgag 1740 tttctctagg gcgccccagg tgagctcgga ctcttatggc ttgatggggt tcattatact 1800 ggcagaagga gagcgttggt tccggcagat taagtagtga agctggggcg gcagggaagc 1860 cgagaccctc ggccaagagg tgacttggga aagcacaaga gggtcgtcag tctggcaagc 1920 acttaatgtc gttcgtctgt tactcgtccc ctcttctttt caatctgccc ttttcatgtc 1980 tttcttgggg gggcgggggg aagaggatta gagcagatcg gagaggtaat aaggtggatc 2040 acatagggca ttgtaggccc ttgcaataat ttgttttttg actgagtaaa atggggacct 2100 tttggagggt tttgatccac ggagtaacag gatctgacct ggtgtcaaaa ggagcactct 2160 ggctgctggg tcgagaatag actgttcaga aataggggtt agtagaggac agaagcaaag 2220 aatatgtttg gagggtgtta cagtaatcca ggcgagacgt gatggtgcag aggccacgct 2280 agaaatacta tggagtgtgt tgaaagtggt caggttttgt ttgaaggtgg agccagtagg 2340 acttcctgaa tattgagtat aggccaacag caaaagaaga aacaaggatt accccaaggt 2400 tttttggttt gtccaactgg agggtgtgtc atcaactgag atagggaagg ctgagattga 2460 accaggtttc aggtggaaga ataggagtta gtttgggtta tatcggtcta agatgactct 2520 caactcagtg ccgctccaag tatggttcct tccttgggct gtttgttgcc tgtctgtgat 2580 gagataaata caggaagtaa aataaaaatt ttaaaacctt tttcagcaat ctgacatcgc 2640 ccagcatttg tatttttttc attaaaaatt ttttttttct aagtatcaaa ctattagtct 2700 acaatggatg gggacaaaaa ctggcctttt tcttatatgt agactagttt gaggttgtga 2760 taagtaaatt taaaataaga ccagctagcc tcgttctatt tttccagcat tgtttagcct 2820 cttgtgtata tgcatagaga aagcatctgg tggagatctt tctggcctag aattttctaa 2880 gaatttgaat acattgatga cgactgtaaa aacaatgctc tattcctgag gcaacctaaa 2940 gatacctatt tcaggcaact ttttaaagtt gaaatttatt ccacatcacc catctgtact 3000 ttcccaacta cattgttcta tttaactctc cccaggaatt gaactcctga atcgtcttca 3060 gtttccagtt ggccactcag ctttcaatat aatgtaccta ataatctcta aatgtttttg 3120 aagaccttca taaacagctt ctactttctt acaagcttct cattgcccct ccagattcta 3180 ttatgtgtta tacaaaaaaa aaaaaaaaag tcataccgta aagggaaata tatctttggg 3240 atatttataa caaaacgtaa gtcatattgc taacaggaaa ccaggaagga ataattactg 3300 tgtttgtctt tgcagaatga ttagaaaaaa caaccctttt ccagaaatag aaatgaaaac 3360 aaaaattctt tagcaataga gttatttttc aaagaaattg ttgacattaa ttgtagaaac 3420 gtgatgatat atgataataa atttaattac tgtatttcca ttttaatgat aattatcttt 3480 attttttcct cttctttttt ttttttttcc cccccaagca atgggccact tttgctagaa 3540 tatggtatct cttagatggg aaaatgcagc cacctggcaa acttgctgct atggcatcta 3600 taagacttca gggattacat aaacctgtgt accatgcact gagtaagtat tattttagac 3660 ctgtcattaa ccactgtagt tttcatgcca gaatagaatc ttgatgaagt ttaacagggc 3720 tgagtttaag ggcccttttt atttttcctg aggatcctgt attccattct tgctgaaacc 3780 atactagcgc ccaaaactga atttggctta ttcactggaa aaccgtatgg atcagtttac 3840 ccggaacaag cctattttat gccactgccc agcgaaatta ttagtagccc ttcttttcac 3900 actcagaaat aacctgattt gccccatgac ttaaactttc acttttatat atgtcttttc 3960 acgtgatgac atccatataa agagttttta agggaaataa ttttgccctc tcttctaaaa 4020 aatcttttcc cctagaaaaa tatatggtgc tagctaggta tgggaagctt attttcacta 4080 ataagactaa tagacttgct tttctgtatc ctttttccaa aataaaatta aaaagttgtt 4140 ttcgttctag aagatttctg cttacagata gtttttgata gacttcattt accccagcac 4200 ttcatggcca gatccagatg tcagttttta gtcctcatac tggttgactt gtcaacagca 4260 tttgctgcaa ttgatcacat ttttatttag cttttattgt gcaatatatt aaacatacaa 4320 atatgtatat ataaaaatat gtgatatttt aaagaaaatt aaccataaca attataatgg 4380 tgaaatgaat atactcagca ttggtttgtt tccaattttt ttggcattat gttatattac 4440 attaatgttg ctatcaatat tcttgtgcca ttttcttagc acacatggaa gaatttttcc 4500 aaggtatgcc caaaagacag gaattactgg gttttaaggt gcatagcaac tattgagtct 4560 ttatcatgcc agtagcatgc aagagtttcc attactcctc atcttcaata acacttgata 4620 tgttcgttct ttttagtttt gactattctg gtagaaataa aatgcttgct ttgtgatttc 4680 aatttgcatt ttctcgatga caaatatggc tggctgagta tcttaccact tattggcctt 4740 tacagcttcc tgtcctgtga aatgtctgtg catgtctttt gtcaattctt cactgagttg 4800 tttgtccttt gcttttttct tacttttatt acaaaaggtt tcaaactatc agaaaaggtc 4860 aaagaatagt acaatgaatt tctttatact ctccacctaa atacagttaa cattttatca 4920 tatatttttt attcaaccat ttgaaaataa gctgcagatg ccattataat tcccccaaag 4980 tacttcagca tgcatctcct aaaaataaag atactctcta tgtaaccaga atgccattga 5040 cacacgtaag aaaagaaaca gtactttatt tagatggtct catgttcagt gcatttttaa 5100 atttctttgg ttttcccaag tatgtttctt acagctatta tttttttgaa ccaggatgca 5160 acctaggttt atgtattgtc tttagttgtt atgttccttt aatctcttaa tctagaataa 5220 tccttctttt attttcttct cattaacctt ttgaggagtc caggctaatt ttcatctgga 5280 gaactttctt cttgaaacac attcttcact tgtttccctg gattcacttg tttccctgtt 5340 tcttctccct ttcttagtac ccttggctat cctcagcctt ctaaactctc aaatgctctt 5400 ctcaccaaaa attggaagtg cgtcagggcc cagtccttga acctcttctc atgccttaga 5460 tgattgcatt ttgtctcatg tatctaaaaa tcatctatat gctaatgcct cccaaattta 5520 aatctccagt tttgactgct tccctgtatt ctagactcac atatacacat ttctattaag 5580 caaattcatc tgttgtctag tagacatctc caatgaataa gtccatttgg agatgtcttg 5640 cttcttatct acttcaccca aaccaacctg ttccttccca gttttccctg tctcaattaa 5700 tagcatcact gttctttttg ttgctctggt ttgaaggctt tgccttatct gtgactcctc 5760 tttgttcaac caaaaaaata aaaaaagtac atatatatat acactcacac acacacacac 5820 acatatttaa aaatatatta gaaaaaagta tatctgaact ctggcacgtc tcatcatctc 5880 tccctctact ctgattttaa gctgctgtac tccatcctgt tttattccaa aaactgcctc 5940 actgtgatct gttttcttct gccattgcct ccacgtaatt ctttctccgt agatcaacta 6000 cagttatctt ttccaaatga aatttctgtg tgtcctacaa gaacctaaat aatatggtcc 6060 ctgttacctt tctgactttc tttcctttat ttatttattt attattattt tatttatttt 6120 tttactatta attgtttctg tcaatccact cagacctata ctgttccatt aacttgcctg 6180 ccgagcacat ttttgcctta tgcctttttt ttttttttga gagagagaga gtctcacttt 6240 gttgcccagg ctagagtgca gtggcccaat cttggctcac tgcaacctcc gcctcctggg 6300 ttcaagcatt tctcctgctt tagcctcccg agaagccggg attacaggca tatgccacca 6360 cacctgacta agtttgtatt tttagtagag acagggtttc accatgttgc caggctggtc 6420 tggaactcct gaccgcaggt gatccaccca ccttggcctc ccaaagtgct tacaggcgtg 6480 agccaccatg cccagccctt aggccctttt cacacactgt tccctctgcc tagatattct 6540 ttaccagata tctgcatagc ttatttcttc atgtctttca gatttctgct caggcattta 6600 tattctgaga gaggcctcgc ctgattcctc tgctcaccct ttcactctat atacctttac 6660 cttgctttaa aaaaaatagt gttcatgtat cacctgaaat gttatgtaag tatctgttta 6720 ttatctacct tctttcacta gactaagttc caagagcaca taaaattttg ccggttttgt 6780 acacagctgt atccccagta cctgtagtag tgcttcacac atagttggaa tgtaagaaat 6840 gtttgttaag tgaatactgg catttatgaa ttccagttgt gaaaatgaag aaaatacttt 6900 tagtcagggc tctggcattg aagctaagtc agatttttgg atctatggag ctaagaggaa 6960 gagtattcca aatgatttga tttgagcaag agcaaggatt ttctgtaatt tcttatactt 7020 gctgctctaa ccttttttct ctacctaatg aaaatttcaa taagatatca aaataaaccc 7080 atactctgaa atctgtgagt atagaatatg aatctgaggt tttgtctttt taatctttgt 7140 ttttcaagca cccagtacaa tgtctggccc ataatagatg cttgccagaa atcatttgta 7200 gaatgacact ttcaaacaga tgttttgtga gtagcctgtt cttgttccct gtagttatcc 7260 tgtgaacact ggattagtga ataccgaacc attgcttctg gggggaaata cggagttagg 7320 ttcctctgaa cctctggtca cagcattttc atcaaccaat caatacataa ccttgtttta 7380 tgtgtgtttc tgtttaaaga tatcttaaca ctgaacttgt gaccaacagc agtataataa 7440 caagcttatc taacacacat attatctcta taaggcaaca acacagcctt cttgtattta 7500 ggaacactaa atagcactca gtataccaag cttgggggat attttaaaca gtgaagtcac 7560 taagaaaaag tccacagatg ccagaaacat agcactacat agaaaatgaa aaggatgctt 7620 gtttgttgta tgaaaggtga aagaaggcag agcatcacct tgttcagcct cagctgggaa 7680 catgtgcgtc aagtaacaca aatttatttc cactctgagc atgtccatga atgactgcaa 7740 aagcactatg agtattgatt ttggggtaac aaataaattt taacaagtag gcaaatttgt 7800 aaatatggaa tctatgaata aagagatcaa ctgtattttg aaagtaaata gtgatcagga 7860 gacttacact ttcacttata aacctgttta ctttaaaaat attttttggc agaccagaaa 7920 aatagtcata gaattaccga tttatagagt tgtttaattc cttaggactt tagatatttt 7980 gtttaaccac ctcatctctt cattgacagt cgagtaaact aaaatcagga tttcattgct 8040 attcagctat attatagtag aactgggact agaacttagg cacctgtttc ttatgtagga 8100 aattccccat tccacttcag tctggtgatt cttaaccact tgtactgagt tttcctgctg 8160 cttttttcct tagcaatggt atcagcagat aataaatgag atctcttagg ttgtctttgc 8220 agaaatgcaa ataatacaaa atttgaggaa aagtgcaatt agtagcagtc acctaatttc 8280 catctccaaa tatagcacta actgtccact caaagttctg cttccttata accaggtcat 8340 cattgcaaaa gcatacctta gatatggatg aataggcatt cggctaggat accagaagag 8400 ctgttgtggc tgcctattag gtttctgtga aagtagaaac ttacttttta aagactgaat 8460 tgcatttttg tttcactatg tgttttgaga cagggtctta ctctgccatc caggctgcag 8520 tgcaatggag ccatcacagc tcagtgcagc ctccaccttc tgagcctccc aggtagctgg 8580 gactataggc gtgtaccatc atacccagct aagtttcttg tagttttcgt agagacatgg 8640 tttcacaatg ttgcacaagc cggtcttact cctgagctca agggatccac tcacctcagc 8700 ctcacaaagt gctggaatta cagatgtgag ccaccttgcc tgggcatgtt tcagtgtatt 8760 taatatgtaa catattagca aaacttctcg tgtccagaag atgtcactaa aatcaaaact 8820 taaattgcaa acaattttca atttataaat atgttgtgtt ttaagagttc attctaacat 8880 ttagtggttt aataagcatt tattgagcaa atattatggg cttataatca ctataaacat 8940 attttttttg aaaattgacc tacagtttcc cagaaaaata atattacaaa tgatgatcat 9000 tgttctacaa atggttgtaa aaattaacaa ataatgtact gaacataatg tatacaatat 9060 ttgaactacc ttatttgtca aatggatgtc tgtgaagtgt tgtggaaacc gacttttcat 9120 tataaggtta ttttttatgg aaaatgtggg ttaaggtaga aaggatgtgg aactggtaaa 9180 ggaatccctc cttctaatta gagctgccct cttgaatttg ggacttgaga actttttgaa 9240 cgtgtttctg ataaatcaaa aaataggtta tatatggtga ttagtggagt atgacttatt 9300 ctttatgagg attctactcc tttcactgcc aactgaatta atgaattaat attcattgag 9360 gaggactata ggagtattta aaggaagcag aggaaaacct ttctttagct ttgctaaaat 9420 tagattatgt taaaatattt ctgttctgca ttgaatgaaa caaaagttgc actcagttta 9480 taaaaattaa tgctatgaat gctaacatac caaacaattc aacagagatc agatttatga 9540 tctctagtat tctgccttgt aagtttatga tgctgttctt aaagttgctt agctaacatt 9600 tttctcttac tattcagtta aatgtaaaaa tgtatttcaa atgttaatat cacagcattt 9660 taattttgca atctaaattg tacatttata cttcacctca ctcctagaat atcatcctac 9720 aaagcactga aggagatttt tgcttcaggc tatatctaga atacgtatct cctttggaag 9780 tgactatatt gatattgtca ataaattaaa caatatgcct tggtgcatgt tacagtatct 9840 atacattcag cagattttat ggtaacctat ctgatcattc atcaccttgg ctgtgcacaa 9900 gttcagggat gccaaaaata acactgaagt agccatttgg acatttgtcc ttccaaactt 9960 agaaacaaac ataaaggcca aagccaaact ttgttgttta tcactaattc agaatagcta 10020 tttttcaaac atttctcaaa ctaaatcttc aaggaaaccc aataaataaa agcagtactg 10080 tcctagttga aggaaaaaga acctggatcc ttgtgccttg agtcaccttt tagaagccta 10140 agactgaatc acctgcactt taaaaaccac tatgctaaat caaacagctt ataaggaagg 10200 attcaaccct ataaagggga agaaaacaag ctacagactg gaagaaaata tttgcaaacc 10260 acatcatcaa caaaagacat atctaaacta taaagaactt tcaaaactca acagtaaaaa 10320 aacaaacaat ctaattaggc tatgggcaga atacatgaac agatggcaga taagtacatg 10380 aaaagatgtt caatataagt agccattagg gaaatgcaaa ttaaaaccgc agtgagataa 10440 tactacacaa ctatcaaaat aactaaaata aaaaatagtg acaacagatg ctgatgagga 10500 tgcatagaaa ctggattact cagcattgct ggtagaaatg taacatggta cagcaactct 10560 ggaaaagaat atggcagttt cttctaaaac taaacatgag cttactgtat agcacagcag 10620 tttcatttct cttggacatt atcccagaga aatgaaaatt aagttcatga aaaacctgta 10680 ccaattaatc gcttcaccct attactcttt atcttcccat gttcatcagc tcctggcata 10740 ttgcatattc atttatttgt ttgtttattg tctggctttt caactagagc ataagattga 10800 ttaacaagaa tttctttgta aggaacttaa acaaatttac aagcaaaaaa caaccctata 10860 aaaaagtggg caaaggacat acataaagag acacttcaaa agaagactta catacagtca 10920 acaagcaaat gaaaagatgc ccatatcgct aatgattaga gaagtgtaag tcaaaaccac 10980 aatgagatac catctcacac cagtcagaat ggctatcatt aaaaggtcaa aaaataatag 11040 acattggcaa ggctgaggag aaaagggcat gcttttacac tgctggtggg agtgcaaatt 11100 agttcagtca ttctggaaag nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 11220 caacccccat gacatgcaat ttacctatat aacacacctg tacatttacc cctgaaccta 11280 aaataaaagt ttaaaaaaat agaggttttt cagttacatt cattgctgta ttcccaacat 11340 caagaatggt acatggcatt agtaagtatg taatacatag caagtggatt tgttcatcaa 11400 tggaaaagga ttataaagca aattttaaaa gagctggaga ctctcagcaa gctgaattgg 11460 tgtctctatc tgaatctgcc cttgcatgga atctgagtat ctcaaacata gttaaaaatg 11520 aacactaggg atttttgtaa acacttattg tgtctgataa ctctatgtga gggatctata 11580 attaagagtc tgcagcaagg atctaggacc aaaaaaatta tacaaatgct agaacaaagt 11640 agagtttgcc atttcagtgg caaaaactgc aaatagtttt gcaccaacct aaatagcatg 11700 gcttacctgg cccagacagt agtgttggaa aaactggtaa ataaggctac aaaaagttat 11760 cctatactct aggtctcctt gaatccctaa tcaaggagct ggtacatggg gaagtacaaa 11820 gatggcaaat aactgtaacc tgtgagcagt ttgctgagaa ggacttctga gatcttagaa 11880 tttagcagaa aagaatacgt gatcaattag tgaatgatat cattgtctcc ttatggcatt 11940 ttaactacca ctttttgcca tctggttgtg gtagaaatgg taagatctgc ctttgaagtt 12000 ttactggagt tgtatttgaa acttaacttt accacttatt agccatataa ttttagctga 12060 gttgaacctc tgactctgtt ttctccgtaa acattattaa atagtaaaag tggtagcagg 12120 cagagggagt acgttagctt tacctaataa gatagttgtg agactttaag tgttaaaagt 12180 gagtataaaa tgcctagctc agtgctttgc atgtagaaga tgttgaataa ttattacttc 12240 ctttcctccc aggcaatttt gaggaaaaat tatgacttca ttctggctta agggagaagg 12300 tgggccaaaa cattgaggcg gtgtaggact aagagctgcc cctgatggtg taacctatct 12360 gagaccttag gtttcctgac atgaagctgt tgagttttca ctttgtctga attagtttga 12420 atttatttgg ccatttaatt gagcacctag tattatactg aggagtcaga taaagttaga 12480 agtttaaggt tagaattttt gtaaataaac tcacaattcc tatttctaaa aattgcttac 12540 tatttttcaa cttaatttct cttcagaaca tctcttgatc aagcactaga gctatgattt 12600 atttacattt ggtacatata tattaaatgt gcagttttaa attaaaaatt ataaaatgaa 12660 actatatgta tttaacttaa tagaaattta gacagaaaca actgtgctat atccagggtt 12720 acagtcttac agtaatactg actttagtgt taatgaaata cagttaaaag catgtattta 12780 attgctggtt agtttttgtt tagagttagt agagaaatgc ttcttagaat gaatttacta 12840 gagatttggc atatattatt gaggaagaac aatttttccc agattagatg tgatacctta 12900 gaactgaaag aatatgattt tcagttgctt tgtggcaatc tgtctaatgc cccttggaag 12960 atcttttttg ctctctgtag atattttttg agtggctcga gatgctttac ctatatttaa 13020 aacttaacaa tttaaaaata aattcagttt taatggaaag tttaaagatt ccattttttc 13080 ctccaaatag atgttatgga aagagcctac accgggtgtt aggtaactta gcgtgtgagc 13140 ctctgcaaat cacttaatct tcttctgctg taaatgtctg aatgtgagct aaagtaattt 13200 cagtgttttt tgaggttgtc tggttatctt cccaccttct ttatttgctg ttctgctgca 13260 ttcgcccaac tgcctcagta gccaacaggg tgtcactttt aggaacttca accatttaat 13320 taaacattgt cagactattc ttttccatct aataacagca agcttcccag gtctttcatt 13380 catacctctg aatttccttt cactctagtg atgaaaaccc acttgcatta tcttcttttc 13440 tttattctta tcctatatag actatgaacg tctgtgaaaa ttctgccact caaatttagg 13500 gctctgcata gaatcttata atctttagtt ttcatatata ttgtacttgt atttcttatt 13560 tttagtactt ttttcttgag cttctagaat atcttggttt gttttttcta ttagtaaaat 13620 aggtatttta taaatgtttc taattacttg attttgttat gtatagtcag ttatttgaaa 13680 atgtacatgt taacttttta gtttgttttt agatgaacag ccaaacttag ggatagaaaa 13740 tgaagcagat acacatattt tgattttcag gtcatctgtc ctgtcaaata tatttacgta 13800 gtcatagttg agagtagcag tttaaattta ctataaacat ggttggaaag aaacctttcc 13860 atggtagtta ataagatcaa agcagtctgt tccctaaatt tgcaaaaaat aataattgta 13920 atgttttaca tatgttttca atttacctgt aggtaaaaca tactttttta tatctatata 13980 catactgttt tcattttctt ggctattttt cagggaaatt ttgtatcaga catttaaagg 14040 cacttaatac aaacattttt actgaagtgt aattattaat gggaaaagtc acaagaataa 14100 cagtaatttg cctttaaaag atgtattgtt gttattggca accttattaa atttctttta 14160 ctttgcagtg ttgagatttt ccaaatggcc agccaactct ttctatactt agatgttata 14220 ttaaaaagta acagaaatct attcaaggat ttttgatgtt gcactgtaac ttgcttttta 14280 ttttttaata tgttgtttga actttgataa gctttataaa taaccaaggc cactttttct 14340 taaggttttt atttttctta caaaatcttg tttcttttta aaatgtaggt gactgtgggg 14400 atcatgttgt tataatgaac acaagacaca ttgcattttc tggaaacaaa tgggaacaaa 14460 aagtatactc ttcgcatact gggtaagata atgctgttgt cagggaaatt aggtagttaa 14520 aatatattac tttaagaata cagttgttct tacttaaaag aaaaaagata tttttaaaaa 14580 tcagcattta ttactaattt tactcatgcc atgtatataa tttactcaat ttttaaaaac 14640 cacacaacta taccaaatat agctattacc tgacagtatt tgtttatcct acaagtgttt 14700 catataacct gtggacaaga ttattctcat gttttagtca ctcttttact ttgtatttct 14760 gcttcctgta attcatctta ctgctgtttt cagaaccatc ccaaaccatc tgaaagttaa 14820 caagatgttt aaggaatggt cactcttctc tagataaaat cttgtttgtc agtatctctc 14880 ttaagtaaat acactcacct gggtgtgttc tgctggtttc caaatacaac gtgactctcc 14940 tccatttttc agttaggcaa tcttctgttg caatagaact tttcttcaag gccatagtat 15000 ttctgtattt gagtttactg tcagctaaaa ctcctaagtc tttttcaggt atattcctgt 15060 tgaatcatgt cttctccttc ctgtgtgctt tagttgtaat ttaattcaat tgcaagacct 15120 taagtttact ccatgttaaa ttttatctcc agccttttaa gatcctgatt ttatcatctc 15180 atgtattagt taacccatag cttcaggtca tgtgcacatt taattaagtg tactttctat 15240 actgttactc aagttatggt tagaaatgtt atcttttatt cttgatcaaa atcactattt 15300 tatttccaac tgcatcttcg cctttgatgt tcccatttgt tacttttcat ctctttcccc 15360 aagtgcatct taaatatttg gaacaactgt tttgcttcct tgagccagcc tttgcccatt 15420 cctcccctta aaatacattc atgaaatctt tatataattt aacaaaactg aatttacatc 15480 cgtctttagt aatttcagct attggcagat ctctatgtat aaaccaagtt ctacaaaatt 15540 gagaggtaat tgggaaagaa atattaaaaa tcagaataac ataatgtgat aaaagcataa 15600 agggaagtat tgacacaaaa tgctatggta attactatag caataatatg tagtattact 15660 gcaaatgaaa gaaaatgaca gcacagccag tagtgaacag ctaccataat gtgtacgtca 15720 gctggtagtg tccctgtcag tagtgctttc tcactgatag gtgaaaattc aaactccttt 15780 acaagtcatt ggaaaccagt tcccactgct gcttcatagt tcttcttaga caacatattg 15840 ctatttcatt cctttctgcc tttgcatgtg ttgttcccct gacagtgagg aattctccta 15900 tttatccatt aaaaattgac tcagatgtca cagtttctgg gatacctttg ctatttcctc 15960 atcgctcaaa gagttgattg ctttttgttt ctgttgactc ttcacttaca ctttggacat 16020 actactttat tatgctcata acactgtatt acaattacat attcaaaatt tagcttgcta 16080 gttgtaaaat ttttaggaca tactaatttt agaaaatata gagaagttta aagagggaat 16140 taaaattcac tcctgatccc atgacttacc attttaatat acagtcagcc ctccatatct 16200 atggattcca cattgtggaa atgtaaaatt ttcattttgt tttgtttttt gagatgaagt 16260 cttgctctgt catcctggct ggagtgcagt ggtacgatct tggctcactg caacctccgc 16320 ctcccaggtt caagcaattc tcctgcctca gcctcctgag tagctgggac tacaggtgtg 16380 caccaccatg cccagctaat ttttgtattt ttagtaggga cagggtttct ccatgttggc 16440 cagactggtc tcgaactcct gacatcaggt gatctgcctg ccttggcctc ccaaagtgct 16500 gtgattacag gcgtgagcca ctgcacccgg cgtgtaattt ttttctattt aattgtgtca 16560 atactgaaca tgtacagacg tttttcttgt cattattccc taaacaatat agtataacaa 16620 ctatttacat agcatttaca ttatattggg tattataagt aatctagaga tgatttgaag 16680 tatacaagag gatgtgtgta ggttatatgc aattactatg ccattttata tcagggatat 16740 gaacatccct ggaatttggt atgctcagga ggtcctggaa ctagtcctcc acagatggag 16800 agggataact gtatatacaa gtttatatcc tttttcactt aatagaaaat tctcagagaa 16860 cctttttgta agtattattt ttggttacta agtagtagtg cacaaaatga tttacgatac 16920 ttgatcattg ttctgttttt tgaaatgaaa attttggctt ttataagggg ctgataaaca 16980 tctttttttt caattcttgt tttttgtttt ttttttttaa cttttaagtt cagttacatg 17040 tgcaggtttg ttacataagt aaacttgtat catgggggtt tgttgtacag gttatttcat 17100 cacccaggta ttaagcccag tacccattag ttattttcct ctctctactc ccaccctcca 17160 ccctccaata ggcctcagtg tgtgttgttc ccctctatgt gtacttgtgt tttcatcatt 17220 gagcttccac ttataagtga gaatatgcag tatttggttt tctgttcttg tgttagtttg 17280 cttagaataa tagcctccag ctgcatccat gttgctgcaa aggacatgat cttgttcttt 17340 tttttatggc tatgtagtat ttcatggtat atatgtacta cattttcttt atccaatcta 17400 cctttgatgg gcatgtaggt taattccatg tctttactat aatgaatagt gctgcagtga 17460 acacacatgt ggatgtgtct tcataataga acaatttata tttcttaggg tatgtaccta 17520 ggaatgggat tgctgggtca aatggtattt ctgtctgtag gactttgagg aatcgccaca 17580 ctgtcttcca cagtggttga acactcccac caacagtgta aaattcccac caacagtgta 17640 aaagcattcc tttttctcta cagccttgcc agcatctgtt attttttgac tttttaatag 17700 tagccattct cactggtgtg agatggcatc tcattgtggt tttgatttgc atttctctaa 17760 tgatcagtga ttttgagctt tttttcatga ttgttggcca tatgtacgtc ttcttttgga 17820 aagggtatgt tcctgtcctt tgcccacttt ttaatggggt ttgttttttc ttcttgtaaa 17880 tttaagttcc ttatgatgct ggatattaga cctttgtcag atgcatagtt tgcaaaaatt 17940 ttctcccatt ctgtaggtgt ctgtttattc tgttagtttc ttttgttgtg cagaagctct 18000 ttagtttaat tacatcccat ctgtcaattt atgcttttgt tgcaattgca gtttgcatct 18060 ttgtcatgaa atctttgccc atgcctgtgt tcttaatgat attgcctagg ttgtcttcca 18120 gggtttttat agttttgggt tttacagtta agtctggaat ccatcctgag ttaatttttg 18180 tatgtggtat aaggaagggg tccagtttca atcttctgca tatggctagc cagttctccc 18240 agcaccattt attgaacagg gaatcctttc tccattgctt ttttggccag gtttgtcaaa 18300 gatcaaatag ttgtaggtgt gtggtcttat ttctgggttt ttttccactg gtgtatgtgt 18360 ctggtttttt tttttttttt tttttatcag taccatgctg ttttggttac tgtagccctg 18420 tagtatagtt tgaagctggg tagcgtgatg cctccagctt tgttcttttt gtttaggatc 18480 gccttggcta ttcaggctct tttttggttt catatgaatt ttgaaatatt tttttctagt 18540 tctgtgaaga gtctcagtgg tagtttaata ggaatagcat tgaatctata aattgctttg 18600 ggcagtatgg ccattttaac aatactgatt cttcctatcc atgagcatgg agtgtttttc 18660 cgtttgtctg tgtcatctct gattgctttt tattattaaa gttttttttc ttttgagatg 18720 ggggctcact gtgttgccca ggctggtctt gaacttgtgg gctcaagaag tcctcacacc 18780 tcagccttcc acagtactgg gattacaggc atgagccatc atacccagcc ttcatctctg 18840 gtttctttgg gcagtagttt gtagttctca ttatagagat ctttcacctc cctaattagc 18900 tgtattccta agtattttat tctttttgtg gcagttgtga atgggagtac attcctgatt 18960 tgactctccg cttgactgtt gctggtgtct aggaatgtta gtgacttttt gctagtgatt 19020 tttgcacatt gattttgtat cctgtgatgt ctttgtccat tgattttttt catatatgta 19080 ggattattta aaataaatcc ctggaagtgt aatgtgtttt gatatcaaag gaatgatcat 19140 ttataggtct tttgataggt attttcaatc atgagttttc taatccatag aagagcatac 19200 aaatgtttgt tatcccaaat ctgtgcaggc acagtattat ctttaaaaat aaggctgttg 19260 tcttttgata tgcagctgaa tatttacatt tacttaataa tgaagtttca gtgttacttt 19320 atcactttct atggacgttt tatctattaa gattattaat ccacttttgt tttttacttt 19380 taacaaaatt tttatgtcat ttactgtagt tatttaattt tatattatta catgcctttt 19440 cctttgtaat ttcttctgtt ttccagtctc agaaattctt tcttgttcag attttcataa 19500 acatttactt ctaatttttt aggggttggt tgaattgggt gtgtgtgcac cttgaacttt 19560 cacatctaag tctttagtca tctaaaatac attggcatga gttagagata caacttaaca 19620 actagttgta tcaacagaat tgtttaattt taaataggac aggattttgt catatttatc 19680 tgtgtgtccc cagttccagc atgatacaca tttaagtgtt tattgaatta atttcaatga 19740 aaaacccttg gctagcattt aaaacaaatg ttctcttact ccatcccata ttggaggaaa 19800 gaatgagtta ttttcttact agtaaaaatc aattgagtgt tgcattactc aagtcatcct 19860 ccactcaaaa ttttacatta gctttccaaa tacgtcagaa taaagtccag acttctgaaa 19920 tatattgtaa atggtatgtt ttagaatttg ccatctcaat tttcaaacta gaaagatcat 19980 ctttcagcat agaaattgta aaatattttt ttccatggag agaaagaatg caggaataga 20040 agtctagaca ggcatttctt tttctaaaac tgaaattcca tgaaaaaaga gaatatcaat 20100 tttaaaacta cccatctttc tcagcaaact tctaactttg acaagtttta ttcctaaaat 20160 gtgttttctt aaatcatctt taaaatattt tcaaatttta ttgaagggaa catatggaaa 20220 aaatcacctt taggtaatct taaggttttt tgcagattcc acactgactt tccaatttct 20280 tccccctttc ttgaaaatcg tttggacttc atctgtttag gaaaatagaa ccttacatct 20340 atatatgtat tatttttgtt ttaaatttcc caatcccttt cacaaatatt ttttcatttg 20400 acccagaaat cttttgaggt agatagcatt atttattcat atgagtaaaa ttatatgcag 20460 agaggtaaaa tgagttgcct gatattacac tgggagtcac tgacaaaacc tagattccat 20520 acctgcatct agggtatctg gaagctaagc tgcctccagt actattcaat gtatattttg 20580 taattgccac tttttttcat tttataaaga ataatcgatc tgaacacaat gaaatattat 20640 attacttaga gccaatatgt agcacttaga gacctttaca aactgctaag aagaaacatg 20700 attactggct gtctagagta tgaacttaat tcagaaccaa gtggactacc aacattttcc 20760 ccagacctaa cccgtagttt atacggttta tctaatttgg taaggaacat gaaatagtct 20820 ggcttgtttc tatttctttg ttaaatcaag tagtaataaa taccccataa tgagtgagtg 20880 aattaataac ttacaattca gatagaaata tacttatgcc aataaatttt tccaagaagt 20940 agaacttttt atgatataat ggtagttgac catttaagag aatagcattt caaaaagcgt 21000 agattactac ttaattttga aaacactaat gaaaagcagt ttcgagaaac aatttattaa 21060 aatagttttc ttataaaaga atgatcagtt taagaataca gtatcagcct atgcccagtt 21120 ttaatgtctt ttctcctttt aacagctacc caggtggatt tagacaagta acagctgctc 21180 agcttcacct gagggatcca gtggcagtaa gtttcttgta ttttttatta atcatctttg 21240 gaaagaccaa ttcagatact gttttgtcag tctcccttct cattatttta atatagttag 21300 cattcaagga tataaattct tcactttagc tgtatcacac acgttttgat aagtattatt 21360 tttaatattc aactctatgt attttctaat tgctatgtat tatgttttgc atttagtcga 21420 agaattctga tgtttttctt tttaattttt gatacctgca taatcaccta gtatatgatg 21480 agtttgctga aatgtcttgg aaaggagaga gatatatgta tgtatgtgtg tacatatatt 21540 ttcaaacatg ctaagtgtgt tcttcttata ttctaacttt tttgttaact cgagtattat 21600 ctgagtttat ttttgtttat taacatctca attgtagatt aatttcttct tacaggtctg 21660 tccattttat tctggaaaaa atagtaaagt tcttaaaaac aaaactactt tcattttatt 21720 ttattattta tttatttatt tttctggaga caaggtctca ctctgtcacc caggctagag 21780 cacagtggca caatgatagc tcactgcagc ctcaaactcc tgggctcaag ggatcttccc 21840 acctcagcct ctcaagcagc tgggactaca ggtacacacc accacaccct gctaaaactt 21900 tttatttttt gtggagacca ggttttccta tgttgcccaa gcttgtctca aactcatggg 21960 ctcaagccat cctcccacct cggcctccca agtgctggga ttacaggcga gagccaccac 22020 actcagactt tactctgttt ttaatttatg tttttaaaga caggatcttg ctatgttgcc 22080 caggctggac tcaaattcct gggctcaagc aatcttccta ttttggcctc ccaaacacct 22140 gggactatag gcaccctgct tgtttatgtt ttatttattt tttatttttt ttcctgtcaa 22200 tttttgcctt atatattttg aaggtttagt gttagatgaa tttaagattg gaattggaat 22260 ctttctaaac aattgatcag ggtcaatatg tactgatcct cttaatctcc aataatgctt 22320 tttaccttga ggtctatttg ttctgacatt aatatagcta cattagctga cttttgatta 22380 gtgtttgcct catatatttc attttttaca ctttactaag ttattctgtt atagatgttt 22440 ttattgtaaa catcatgtaa ctagattttt ttttctctcc tttcttgctg ccttttggat 22500 tggttgaatt gatttattat agctctaagg ttcatcagta tttttacctt acttccaaat 22560 caatgaaagc ccttagaaga tttttaacta ccaccctccc tcccaatcta caagctttct 22620 ttgttcaggt ttttaaaaaa actcttccaa ttgaacattc ttgttattgt tttattctgt 22680 tactgatttt tttaaatttt cttttcttac aatcccttct tgtgactcag cctgccttct 22740 ggtatcaatt tccttttcct tgaagtactt cttgctggca ttcttcaagc cacattctct 22800 taatactgaa ctcacctttt ttgtctacaa ctgttttctt tattttgtgc tttcattctt 22860 gaaagatagt tttgctatat gtggaattct aagataacaa ttattccatt atttttccct 22920 cagctttttg aagctattat tcaattggaa tttagcttgt attattgctt ttatgaatca 22980 gatgtcgatt taattgtttc tttatgtgta ctcttgtctt ttttttctgg atacctttaa 23040 gaccttttca ttgtctttgg ttttcatatc ttcctactac agtcttattt atttggtttg 23100 agtactggtg tcttcagttc tggaaatgtg ttagctctta aatcttctaa tattgggtct 23160 ctttattctc tgtttcttcc ctttggaaat ctggttataa taagagtaga cagttgcatt 23220 tgattgtaca tgattcagta cctctctttc atacttcatg tctcactgtc tctgtggggc 23280 attctcagta atttcttcag atctgtgttc cagttctgta tcttttcacc tgtgtctaat 23340 ctgattgacc tttttctgag gtgctaattt caattatcat ttttcccttt cttaaagttt 23400 tattatttgg ttcttttaca aatttgttga tcagtttcaa tagtcttctg tttatcatgt 23460 tttggttcac tcttatattt ctttatgcat actaaacata attcttagag tttagatctg 23520 attctaatat ttgtggcctt tgtaggtccg caatttataa tatctgctca gtgtacttac 23580 ttggtaggct catgtacctt gaaacttcat ctccaggaat tctttaagac ctgtgttaaa 23640 aatgactttc ctcaaaaaag attcacttag cttatggcta agtgaattgg gctataacaa 23700 cctaagtttt ttgagctgaa ctttagactt acaggatttt gatccccaca agaaggatga 23760 atttggggcc tcaaagcctt gtgaggtcag ctttgtttca tactttaaaa aatattttta 23820 taaacaatat taattaactt cacgaattaa ctaaattttt aattactttt aattttaact 23880 acataaatat ataagtttat atatatttat atgtttatat atgtttatat atatgtttat 23940 atatatattt ccatatatta aatgacctct cagaaacata aagtcttaac actaatagaa 24000 aacagatgga aaatttatat ggttcattaa tgtttatttc tttgtgtatt atactgggag 24060 aagttctaat tcatacatta gtcaccaata ttgctaaagg caaagtctcc tgttgttttt 24120 tcttttagct attgatagac ctaaactttc aactttactc ataggttaga tagcatgttt 24180 ttcctattag agactatttt atctttttgc tgcaatacat taataaatta gcaaagtgct 24240 agacctaaaa atatcatttt ttgtttccct aaaataataa aaataaatat ctattgtcag 24300 gatacaacca tttgtttggt ataatactgc ctaaaatttt cttggtaagg ttggggggaa 24360 ggatatttta ttataataag acctgacata ccatttaaaa gtttcatgat ttactattta 24420 ttttgtattg tttatatctg ttttataaaa ctgtgctcag aatttgcttc ttaggaaata 24480 tttaattttt ttttcataac tagtgtgttt ttaagcctta caggtcaaaa tattatattt 24540 tgaatattaa tatgctttgt atataatagc attaaaagtt ataaggctcc tgataatata 24600 tatcacagct gttaacaata aattatactt ttaatacttc ataaatattt taaatgttaa 24660 aatgaaagtt tctattttat gttaactgta cactaggagg cagtatagta taactttcat 24720 gttaaaaatt tggtcttagg taacttttgt agaaattgtt tcaaattatt gtttatgagc 24780 aataattgaa atggattgta aaatgtttga ggaaggctta ggaaatattt cagagttaaa 24840 tccatttgga ttattacctc attaaaggat tttaattttt taagtcttaa tttttactat 24900 gttttgaaaa agaatggctc gtcaaggaag agatttatta tttactttta attcaaatcc 24960 taagtttttg tctggaatag taggcctctg ccagtagaca taatttgctt atcagttctt 25020 actgtgtgtg gcctatcgga gaatttgttt cacaatgcag ctttctgatt attagtacag 25080 tattggaata ctatttgtga aatatcttaa ttaatattta atagttaaat aggtaatagt 25140 ttgttaaata tttaaaattt ttcataatga tgtagattat tctaatttca aataaatata 25200 tcgatttagc cctttcctta tgttcttttt ctctctgttg tatgttttag cttctttaag 25260 aggttctatg tccttatatt ttcttctaac atggttcctc tatttgattt ttccccttcc 25320 tataaataat aagttacttc tatggcacaa atggcacgta ccctgggata tagattatat 25380 gaggaaggtc tttgatattt actaacttta tcttattcta ttttttttac tttctgaatt 25440 tcttaaaact tatacttaat gtctgtacag ctagaattac ttttttgcat gtacatgttg 25500 tttcaccttt gtttacgtac atttgtatgt ctgtcttttt tcttccctgt tagcttataa 25560 tcttttcaca ttttctagcc tttttaaatt tgagtgtgga gtttgcattt tctaataggg 25620 cagattttta aaataccaac ttagtaattc aaataaatac aactaaattt tttagaaaca 25680 tgaaaatcaa atgccgtgta acactattaa tacaataaaa tgcctttatg ataatattca 25740 gaggatataa ttacttgctt atccagaaaa gatagtctta acattgtcag tttcaattta 25800 caacttttaa aacattgaac aatttcagtg ctaataacta aatcagtgct gtctagtaga 25860 aatttttgcc gtgatggaaa tgttctgtgt ctgcactgtc aaacatggta gccactagct 25920 acatgtcaac cgaccacttg aaatgtggct ttcatgacta aggaactaaa tttttaattg 25980 cttttaattt taacaacata aatatatata tatttatata tattttatat atatttatat 26040 atgtttatat atatgtttat atatatttac atacattaaa tgacctcaaa aacataaagt 26100 cttaacacta atagaaaata gatggaaaat gtatatggta acccattagc ttttgcatgt 26160 aatgaggctt tgtggtctga tttgatgaga gaataaaaga ggaaggcctc ttaacaaagt 26220 aaccctattc taacatgtat tgttttagtg ggtttagctt taacacacat caaattatat 26280 tgaaatatga gtgcaaatga taattattct atctaaaatt ggcacatata cgggttactt 26340 ttaatctttt ttctttttct aaataaacaa taaatggagc caaaagaaaa tagtaatgta 26400 caaagtaata aaagtttctt ttctgttgct tccacatatc cacttaggtc tatttctttc 26460 ctatattcgt gttgaatcta aacccctcat tttcatcatc atctctaatt atgtttgctt 26520 tcttactatc gcaagtcaca attttcttat tgtgtcctat gtatatcttt cagattgtaa 26580 aactagctat ttatggcatg ctgccaaaaa accttcacag aagaacaatg atggaaaggt 26640 tgcatctttt tccagatgag gtaagtcagt ggtcattggt aacaatgctt ttccaaaatt 26700 ttcaattaaa tgtttcattt tctttttaaa taaatgccac agacttattt tcaaaaattt 26760 tatgtcaact tctgaaatat ttttgtattt ttaaatgaat atgtatgtat aaaaaatgat 26820 aattctatta atattttaaa atgtaaaatg aaccatcatt ggactaaaaa ttaaaagtaa 26880 gtttttaaaa aaaattttaa tcactgcttg cttttgaaca aggagctatg atcagaaacc 26940 ctgttaaatt gaggtacttg ttgataattt cattcactat gaaaaagtgt taatttagtt 27000 cactttttat gtgaccagta gcaaaacttc tgtgaatcat ggataaatag cttaactttg 27060 gttgctattg tttcccatga tttaacttta gaatacttgt tcatactcat agttgattca 27120 gctaaaaatt acttggaaaa gagtgtaaac atttttattt attagtatta tattgtttta 27180 agtacttaac ttttctcctt aatatgatgt aatgaaatat taaagcttct ataataaaat 27240 aatattttat caagtcagac tgcctggctt tgaatctgtg ccccattatt tactgactgc 27300 atggacttgg gctcgttact tatctctgtt ttctaatctg tgaagttgac ataataatag 27360 tatctatttt acaagattgt tgtaaggatt aaataaaata cacttgtaga ttgcttagaa 27420 cagaaactgg cacaataagt gctagatgct atcattacca tggttataat tgtccttctt 27480 gttccttctg aaggaatact gtagaaattt attccaaaat atcaagttat aacgattttt 27540 caagctattt atgtgctctt actgtttaag ctttaaacaa tacatgaata gttattaata 27600 atgagtaagt tattcatgct ttatcaaaat gattgattta ttgtcttcgc tataagtgaa 27660 taagtagaat aatttcccac tattcacttt acctctaagg tagcatgtat gggagcagta 27720 tcatcatcca aggatcactt catgatgaaa ttgcaagaat ttaagaactc ctatttctga 27780 ttgtattgtc actttcttta gaaaccataa tttctaaaag acacaaaaat aatatttatt 27840 tcatttacaa gatacgtttg agtgtagata ttttaagtat tcacgatagt tccgttaaat 27900 aggaaattat ttcatttcag actataaaat aaatgagcct agttcccaaa agtgaattgg 27960 taagtattca gaagcttgac agtacatttt atcaagccga tgggggaaac aggcacactt 28020 atacattgtt ggtggtagca caacatggtg catctcatct ggaatcctgg gaattaatta 28080 ggtaacatat aatgaaaata catacatcag gagattgatc cttgatccag caatctcatt 28140 tataaaaata caccttcaag atacacatgc acatgattat tttcactgta ttcaataatg 28200 gtacaataag aaaaacaaat aatgcttatt cataggaaat aaattatcga gtattgtgca 28260 attttttaaa aaaagaatga cagatatttt tgtgacttgg taagttgtac tttccaggat 28320 atattgttaa gtgaaaacaa acaaggtaaa aaagaatgta gatggcatac tacatttgta 28380 tgcatatata cctatgtatg tttacataca cacaagcata tatttatgtg cgctactttt 28440 tgcgtaaagc acgggaagag aaagttgcaa ctactagact atgggtacaa aggaataggg 28500 aaagaatgag atttttttca gagactatct ttaaatttat tttttatgtt aatcatgcta 28560 aaattttata tattagaaaa ataaaatgac agcaacagta gaagtgagca caactagttc 28620 ccaaatcttg ttttccacat gctattccct accaaaagga acttagttct tggagaactg 28680 attgattcac gatctggagc agagagagaa ctaaattatt atagggtata cacatgcaaa 28740 agactattat ttctgcatga tttataatag tgaaataata gaaagtaaca gaaaataagg 28800 caatactcaa aaactaatgg agcaatatca agggaacatg gagccagctt taggtggtgc 28860 ccactgacca aatacaggac tgtgtgagcc tcaaaacaca tggtaataat aagggattac 28920 actgaataag aaaacaaaaa tctatacggc tacagtgacc atccaaaaat tatttttaat 28980 ttattatgat gtcccttctt tctgaggata gaaaagccct ctatagaaga atatcatata 29040 aaaaatatgg aaggaatact gaactagaaa agccatcatt accagcgtat tctgtggcca 29100 ctgggtaaaa ggttattgtg gagcaggata tttatagttt ccaaatacca caatacagag 29160 tatttattat ttgtaaaggg catcttttca attatttgtg atgggaatct tcccattgct 29220 tttacattaa taaaatctgg tggacaccac cgtaaccatg agatcaaatc tttggtcact 29280 gtgggaaaaa ctgacatcag gtgcctcctg ctatgaagca ctgaacactc aacataaact 29340 atatggcctc ttgtcagtaa tgcttaacct gaatgtaatc atggaacaac aatagtataa 29400 atccaaataa aaggaaattc tgcaacataa ctggcctgga ctcttgaaat attaaagaaa 29460 aggcaaggaa acttacgata aaaggagact agactacaga gtaaacaaca taatatagta 29520 actaaatgca tgtgattctt gacctgatcc tgaattggga aaagttacat atgacaatat 29580 tagacctatt tgggaaattt gaatatgtac tgtatattag ataaaaagta tttcatcaat 29640 gttaattttc ctgaacataa aatttaatgt tctataggga gactaggaga tacatgctga 29700 aatatttagt ttgaagtttt tgcaaattac tctaaaatgg ttcatccaaa aagtgtttgt 29760 atatagacag accaaaagag aaagcaaaca tgtcatgcta acagttgggg aacttaggta 29820 aagattagat taatgttcat tatattgtca caacttttgt gtaaatttaa atttttctaa 29880 agtaaaaagt tgcaaaaaaa gtgatcactt gacccctgtt tctgtttgtt gctctggttc 29940 tttccctcat ttccccttag gttaaccacc tctgtgtgaa gggaactctt tatttttatt 30000 tatttattta tttattttga gacggagtct cgctctgtca cccaggctgg agtgcagtgg 30060 cacaatctgg gctcactgca agctccgcct cctaggttca cgccattctc ctgcctcagc 30120 ctcccaagta gctgggacta caggcacccg ccaccacgcc cggctaattt tttgtatttt 30180 tagtagagac ggggtttcac cgtgttagcc aggatggtct cgatctcctg acctcgtgat 30240 ccacccgcct ctgcctccca aagtgctggg attacagacg tgagccaccg tgcccggcca 30300 aagagaactc ttaaatctcc ataaacacat ttagttagca ttcatgggtt accttctctg 30360 tatgcacatt aaaggaggta cttctccctc ttcctggttt catgtgtaat acctaatcaa 30420 aaactttgat tcctcatttt gcattgattt taaccattca gcaaaatcct ctaatagtaa 30480 agaaatttgg ctcattgtat tcaaaattct attccattca gcaaacatta gctaggaagc 30540 cactgtgttc cacatgctct acgctgaatg agcacttagc agctttttga aattgtcttt 30600 acaactgatc tagggaattt tatactggct ttccaaagaa gtatgaaata tttatacatc 30660 tttggaaata tctgacaggt aggcgccttc aataagtaaa aaataagtat caccaacaat 30720 cttttatatg tccaggcatc ctctgcttgc cctgactctg taattattgt gcagatttga 30780 atatactgag aaagtgataa caataggaga aaggatgtta aatattgtta aatgcttttc 30840 agtgaagaag cacattatca agagtatcta ttgtgttttc tgtatgttga tagtgaccca 30900 gcctatccaa accacaattt ttaaaattca tttaaattgc ctaaaatatt ataaatattt 30960 tgctatattg ggttttagaa caagcttaca ggacagtaga gctctgtgat cctaaagtta 31020 taaaatttta tcctgattct taagacaaca tgtcttaaga caacatgtct taagaattga 31080 tcaaagtgct gccttatttg atgccatgtc cttggccaga tattggattc ccacttgtaa 31140 tagtgcgctc tgcagagagc tgtaatttac tgtgtgataa tccttttatc gaaaattcct 31200 gtacacatac tataatgaga agagggaatt atatctactc tccttataag aatgcctata 31260 agacacttcc atactgatat cttaccgtta tttaaaaaga gttcccaaaa tcaatttatc 31320 atccagtctc aatacagttc attctcccag ctgccttatt tctgcatcat gagtacaata 31380 taaatgttca ttcttttggt tacccacact cttcatttta tcattcctcc ttagtctgtc 31440 taattccaat gctttcttct tttaaaatgt cttagatatg tgctttactc tttagtgtgc 31500 ctttcagtat ctaaactcaa actttaaaaa atcttacctt gattactgaa acagccactt 31560 aactcttctt gtctccagct tttctccact gcagttcatt gtgtctactg ttggcaggtc 31620 aatcttccaa ctcttcattt ttctgaatat tggatacata gtagggggaa agtggtcctg 31680 gtcaggggaa tacaattaat aaagatatca tgatttgagt ctagagcaac acatgaaagg 31740 ttgaggaggt tgattagaca gaaaccatgg ttgtgtctgt ggagagtgac attggagagg 31800 tggactagtg gtaacgacac tgtctagcta agcaaattta gcaaaggagt tagagcttta 31860 tcctgaaaaa ttttaagctg agaggtgact ttttaaaaat tataagcctt tagaaggctt 31920 gatcttttta ttttccttta tatggccagc acctggcaca atttttggtt cttagaagtt 31980 caataaatgt ttcttagata aataaatgac tcaaagattt tcattttcaa atttttttct 32040 ggccacagtt ggagcactga ttgaaaggta gtggcactaa acccggtaga cccagtacat 32100 atacttgtca gatgagaaag catgagaatg agccatagca gtgtcttttt gcaattgaaa 32160 agagggaaat aaacagcaca ttttaaaaaa taggtatctt attataatgg gattacttgt 32220 attattaacc taatgtactt ttggggaatt tttataaggt ttttatttta aagattttat 32280 gttttcctta ttctacgagc attcttttgc ttattttttt aacacttatg aagtattagt 32340 aatttattgc taagaaagat attttaagaa aattaagtct aaattttttt ttaatgtagt 32400 atattccaga agatattctt aagaatttag tagaggagct tcctcaacca cgaaaaatac 32460 ctaaacgtct agatgagtac acacaagaag aaatagacgc cttcccaaga ttgtggactc 32520 cgtaagtgtt tcccttcttc ttgttttttt ttcttttgat atcctctgtt tcctcaacca 32580 tatgattaaa taagatcctt gagggcggga agagtgaact catttttttg ctcccctggg 32640 aactcagcag ctactaccta caaataaact ctagatgtag gtattagaac caaaaatttt 32700 taagcattaa gaccttgcta gataagtgcg aggatgttca taggttccta taattatcaa 32760 gtattttgtt tgggctgatg gtagtcttta ggaatgatat atgaattgtt attaatctaa 32820 aacgtaatga ttctcagttt tccacattgt ttcataagtt aaaaatatta tctaatgaaa 32880 agtcattaat tttggatact tgttgtcttg gtccaaaaag aaaaacaaaa taaaaaaata 32940 ctttttgctc tcctctgtac agtttcctct acctaaggcc gtaaagctgc taagatgtaa 33000 cattctttct cccatctagt tgtttctagg cttttttgcc agggctatcc agatgatgaa 33060 gcatttaagt gtaacttcct caggcagtcc ttcctgtaat ctttgaaccg ggttaggctc 33120 tcattatggg ttctcatcgt acacttccat ttactgattt actctaatta tatatttgtg 33180 tgattatctg tttactatct gtctttcttg ctagacttca tgagggagct acatgtttgt 33240 cttatttagc actgagcatg gtgctggaca aattaaatag acactcagcc agtatttatt 33300 gaataaatta attaagactt ttatcagctt ttataagtga ctttaggaaa gaatctctag 33360 aaaatatgaa gcaaagacca cttttcattt ttttcacagc atttatcact atctactgta 33420 ctctcattta tatgtgtgtc tgttatttgt ctctctacac tcatggcaat aataaaaata 33480 gcaagcacta ataaagcatt tattgtgtgc tttacatgta ttaatctact aatcctcaga 33540 gcaatctagt gagtcagtac aatttttatt tttattttta ttttttaaat gagaaaatat 33600 gggcacagag agataaagca acttgactaa tatcacatag ttataaatgg tatattaagg 33660 attcaagcct gagtgactag ctgctgggtc atgctcctaa ctctgctgga atgccccttc 33720 ttgcattggc atgtcagctt caagggggca gggacattgt ctggttcaac atttacatct 33780 tagaaatact tagaaaccct tagaaatagt acttggtgca taatgggcac tcagtaagtg 33840 ttttggaaag aatgaagaca tgagctcttt ccctaaaaat taattgtgaa attttcatga 33900 atagcttaag ttttagaaaa atccaaacca aggttcaaat cttgcctcta tagtagctga 33960 ctctataacc ttaagtaagt catctttcat tttgagcctt agtttcttca tctcaactca 34020 gtttcttcag ttgtaaatta gagccactcc atacttctga gaattgctgg aaggagtaag 34080 tgagcaactt agcacacttg aagtgttgac taagttttta ataggagaat ttttttgcta 34140 ttgcaaattc tataccattt taacatgctt gctagtgtag agttttattg taaatgtact 34200 taagttgatg tagtttgaaa atattcagct ttgataagtt atatactaaa aacattttaa 34260 gttgtccttg aagttttgac aggttacctt cctataacag ttctgtaaat atgtatctga 34320 ttttcttaca gaaacaccat cataaatgaa tttatattcc tgaccaagag gggtgtggta 34380 ctcttttttt taaaattaga aataataaag ttgctgttaa atcatgtatg tgcaaataca 34440 tattatacac ttgaagtcat atttcatgaa tattctaaag tgtattctaa atcagttaaa 34500 ctggcaccac actaatataa taattcacaa attatattta gtgttattat agtcagagta 34560 ctgaccaaaa ttttcattct gctgaatgat ttctgctttc ttttgaaaca aattagaaag 34620 tgcctgagtg ccttcatttt attaatagaa ttagaaaggg cttttctata gctattttta 34680 aggagatttt tagagaaact tagagccact agatgcattt tttagttact actctgggaa 34740 tagattttcc tgccctctct cttcattcca gatttccctg aggcaggaat atgcattaga 34800 cactaggttt tcctttatgc tttattaagg ttgtgatttt ttcctcataa atagtcacac 34860 gcatatgtta acttgtattt tcaaaatagt aaaataggta tacgtttact gaaacgtatg 34920 gatagtatag gtgtactagt catttaatat aaatcattct gtatttcttt ctccctgcac 34980 atttgagctg acatcaagtc cagttagtct ttattacgtg aaaagtgtgt gaccaggaaa 35040 aatatagtca tttttggatt aaccaaaagc ttatctaaga aatagatgat catttacttc 35100 aaggctcttg agtagaactt tctacaatga tgaaaacaac tctcagccag gcatggtggc 35160 tcatgcctat aatcctagca ctttgggagg ccaaggcagg agtacctctt gagttcagga 35220 gttcaagacc ggcctgggca agatggcaag accctgtctc tacaaaaaca aaaaacaaaa 35280 aaattagcca ggtgtgttag cacacacctg tagtcccagc tacttggtag tctgaggcag 35340 gaggatctct tgagcccagg agtttgcagc tgcagtgagc catgatcatg ccactgcact 35400 ccagcctgaa caacagagca agacttgtcg agagaagggg agggagaggg agtgggagag 35460 tgaggagagg gagagggaag cctggcgcgg tggatcacct aaggttggta gttcaagacc 35520 agcctggcca acatggtgaa accccatctc taataaaaat acaaaaaaaa ctagccggat 35580 atggtggtgg gcacctgtaa tcccagctac tctggaggct gaggcaggag aattgcttga 35640 acccgggagg cggaggttgc cgagatcgca ccattgcact ccagcctgcg tgacaagagc 35700 aaaactccat ctcaaaaaaa aaagagagag aaaggaagga aggagggagg gatggaggga 35760 gggaggtaac tctctattcg agcttcaaat acattagcta ctgttgacgt gtggctcttg 35820 agcccttgca ctgtggctaa tgtgactaag gaactgaatt ttttatttga tgtaatttta 35880 gtaaataaaa ttacgtgtta gtgaataaaa ttacgctacc ttcatatggc tagtgtctac 35940 attattggac tatgctattt tagttattgg actatgccat tttataaagt tctattaaat 36000 attatatact ttagaattag aattaaaata tttgatatgc catgcagttc aattcatgga 36060 acatgaggtt tttattatta aaggattgcc tttattagaa aaaaattttt ttctcctttt 36120 ggagaggacc ttgcttaaga tatgcttttg ttctacccaa catagctaag ccaagaatat 36180 aattttcctt tggaatgtag taactggaat ttgtttaaga taaatctgat ggtgaatctg 36240 cagagacttg actgaaacaa atgtacctgc attgcgccac aagtatatag ccacaaaaat 36300 agcttgctct ggtgtttgct gccctagtaa taaaacttaa aatatgttca taatattaga 36360 atgtacatct tataatttga actattatta atatttggtt aaatataagt ttattaactt 36420 ttctattaga gactaaaagc ttctgagcag cagaaacaat tatcaaagag ctttgaaccc 36480 agtagatgat taacaaaatt gctttaatct taattaaaag ctagcattgt tgggatttta 36540 tttaaattat tttaaatcat cttcaaagat tatattcttt caggcctagt ttcagcatac 36600 attaggtttt cttttgctag ttgttgttca gtcattagtt attgtcattt ttaagctatg 36660 gtaggagata aggaatgaaa ttgatattta tgacaaaact atactttttt aaaactaaaa 36720 gccattttat atatactatg ggtactattg cccattcatc tcacttctta agttagtgga 36780 tattcttggt gtattagtat attttacata aatcatggat acataaattt tgtgaaaata 36840 tgttaggaag tttattttat atttcttgac tgtacaaagc agatatgtat ttgcaatgtg 36900 caaatggtga ctgtggggtt tatacttaga ttttttttct tttttcttta aaggaaaaaa 36960 atagcactat cttttcttca caattctgtt gcttaagatt gtagctttaa aatggtcaat 37020 ttattttaat actatcaatt gttacatgat tattgcagtt ttgccaacac atttcacaac 37080 cccaataatc atggaaatta atcatgctca ttgtaaggga aatgtttcaa aatgtagtct 37140 acaaataaaa cttggatcat tttaaacttt attttcaagt tttgcaaaat gatgcaggag 37200 agcttttcct aaggaaaact acgtatgtga aaaaattcaa cttgaattgc tttgcattat 37260 ttttccttac atgttaattc agtaagaaat gcttgtttta agcaaaactc tcacctttgt 37320 agagaagcta accatggaaa gcttcagcat caatacatgc ttgaacacag gtttttataa 37380 cagaaacctg tgttcctggt gtttcagtac tctagagtgt gctattagaa atgtgcctgc 37440 aaaggagaga tggatggtaa ggatgaagac aaggaagtat gaggacaacc attactgatg 37500 cagaactgtc tcttcgaaag taggatgagg attagggagt tcaatatgtg atccattcag 37560 ctctgtaaaa gaaaagctgg attccagatg ctgggaacat tagcactgca ttggtgagca 37620 tcctcttcag tctatagctc tagaagagag cacggattta agcttagtgc atattctctg 37680 atatgaaagc tgttaatcct aaaaactacc cattttcaga tttacttaat actcagaaaa 37740 agattatttt tacctatgcc tttataaaac atgcttcaga tcagagactt aaagacaatg 37800 tagaaggatt tttacctttt ctaatccata agttgctggg atctggacag ctttaagatt 37860 ttccataatt ttcttctagt ctagtcatgt ggcattttct ccacacaatt taacttaatt 37920 tgctgctctt cacagtcagc agttactagc tggacttgtg gactaaggtt actttccaac 37980 tttcctacct atttcaacca gttcagcatt gacttcaatg atagacttca gtctttccag 38040 tcctgggttt tgctcaaatt aggcattcta agctgtactg ttcttttgga tgtgaataga 38100 tcaggactgg ctagtaaaat aaaaaagtag tctctattta aacaacaaaa cccaaatcat 38160 ttataagtcc tagctgatac agattttgac taaaaaagtg cagttgaatg taatggcaat 38220 ttctgttcat tttaattgaa aaaatcattt ggcaaaaatt ttcaacaatc ttacaggttt 38280 agtttattta aaagtatgtt gatctgtagg tctggatctg aaagggaaga aacaaaaaag 38340 aataatcaag aaaggattta aaaaaacatt ttatttaaat gtatgttgag ctttttcgga 38400 tcaggatggt gctacagtgc ctaggaaaat tagtattaag gcaccattac tttacctctc 38460 agtatagcta gcatctttaa agaatatgtg atgtcaccag atttcctgaa atggtttatt 38520 ctaaataatt tcattcattc aatggaagtg acttaaaact caaactgacc ttttatcgct 38580 tttaagatta gttcaccatc caaatattat tttagtcttt taaatgtttc atagaataag 38640 cattatacta agaaatatta tgtaagagtg aaaagacata aaacctattg aagaaaaaaa 38700 tttttttgag atttattaca cttaagctac actcaatttc ctggacttac agctgaaata 38760 gggaattttt ttacttgaaa aataaattat tcttagtagg accaataatg tattttttgt 38820 tttgttttgt tttgttttgt tttgttttgt tttgttttgt tttagatgga gttttgctct 38880 ttttgcccag gctggggtgg tgaaatggcg cgatctcggc tcactgcaac ctctgcctcc 38940 cgggttcaag caattctcct gcctcatcct cccaagtagc tgggattaca ggagcccgcc 39000 accacgccca gctaattttt tgtatttttt gtagagactg ggtttcacca tgttggccag 39060 gatggtctcg atctcttgac ctcatgatct gcccacctcg gcctcccaaa gtgctgggaa 39120 gtaggaccaa taatgttaag tgtcacttat cttaatgtag cataaaccat gtgcttcata 39180 acttgatgtt ttgtttcatt cacaaccaag taaatttttc ctaccatttt ataatacaaa 39240 ctaaaatctc cgaaaagttt tttcctaatg ttaagaggtg agaaattact ccattttcaa 39300 gataatataa atgttttgac ttctgtcatt tctgaattct gaatttgaat ctgctttttt 39360 gatatgtgct tatttcatcc taatgagtag cagtattttg cttgtttcat ctaatgagta 39420 gcaggcagtg ttttgcatag ggatgcgtat ggtaaagaat ttgctatagg aaacagaatg 39480 atgtggcatg tggtgtgatt ttaactgttt ttgagcagtc acaagcacca gagaatattt 39540 tctacaactc agatgaggct ttttaaagtt ctgttttctc actcaaaatg taatactatt 39600 ttcgaaagtt actttgtata aaggcagttt ttgctagtga caagtatctg caaaatagat 39660 agttgttaca gaacatttga tagctatgga aagtttagca caatttagag aatgaattaa 39720 ggaaagtttt atgttagcta tttcttagat gattcattaa tctcccaagt atattttaag 39780 cacataaatg aaaatttaaa actttcgtct ttcacttcaa agatcaaagc ttgtgtgtgt 39840 ttcatatatt tttatatata agtatactat atataaattg tatataaaat atatatcata 39900 gctgtattag ataacaatta cacacacaca cacacacaca cacacacaca cacacatgca 39960 cacatatata atccactgtt agaaactgct ttataaagat gaggactggg tcctgctcac 40020 tactttattc ccggcgccca cagcaggacc tgaccagtca atttgttgaa taaatgaatg 40080 catgaatgga atcatatcct ccctgaaagg gaaattgatg ttcagttcat gaagtgtgat 40140 aaattcatgt tttgggtttt gctccttact taaatcttgt ctaactataa ctaaaataga 40200 catttttctt aacaggcctt actttaatgc aactctaata tatacaattt tggatacttt 40260 gtattacatg tctaaagaac aatcatggta atttcattaa aatacaactt atatttctgt 40320 caaggagtaa atttgtataa tttatatgaa aaaaattgac aggtgttagc tttcagaaat 40380 ggcatttgaa atatccctct aaagatttcc tgtgattatt ttagaatact gttattattt 40440 acaaatatat taaccttata taacattaac gcacatttta aaactacata atttgttatt 40500 tccaatgaaa cagtgacaaa gaagtagggc taatgtacaa tatacattca cctatcatcc 40560 ctcttttaaa agattaagcc gtttcattta agtttcaaat atttaagcat tcatagtatg 40620 aaaagtaatg tgctagatgc cacccttctt gtttttaaaa actatttaat ctagtgaaga 40680 gattaggtca cctcagcaca aaatagggta tgtaagcaac ataatctgag aattcaattc 40740 tagtttgcat gttaccatat atgggttacc aacttttaaa gaacctaaat acatttgctg 40800 agtactaaag ctgcttgaac tacagtttgg gagaaaagaa ccaggaaaga tactgtgtat 40860 caaatggagt catctctagt ttaggaagaa gtatgtgtgt taccttctgt tagatagtat 40920 tagtaatttc ttactgaagg tgctcctctg tcttataaat agctctgtct ttggtctaag 40980 gggaacccat gctaattaga atgtggtcca taaaccagca atgcatcaag tgggaacttg 41040 ttaggattgc caaatcgtga gcccctccac agaataagtg aaatcaggac ctgcattctc 41100 ataagaatcc aagcaatttg tatgcctgct gaagtttgag aaacacagac ataaatagcc 41160 ctctcctccc ctttcttata ttaccttgaa tttcgttgtt ttagtggaaa ggaataagca 41220 atacagaaaa gttgtgtgta tgcttgtttt catttctgtt tgagacagag agaagtaaaa 41280 ccatatttag tgtaaaaacc tctcttttcc ccacagggga acctcctgaa aaatgtgtat 41340 cattaattgc ctccagtatg agttgatgtt tccagctgtg ccactatctt aactatctag 41400 tctttggcca tgatactgct tttccccctt tggaagcaaa ttaaccatct ggaaaatatc 41460 ccgattattt aaagatgaat ttgcacatga agtggcattt tcaagggggt cactgtactc 41520 tgtactgtgc tcagatatac taggcatcca ctgtgtatca ggcattaccg tggctgctgg 41580 gagtataaga taatcatatc agggccctgc cttcaaagga ctggcagcct aagaggcatc 41640 atatagaata aaatggtgct tgcaagcacc ggagtcaaat tgtttacact cagatccctg 41700 ccatctacct cctagctgtg tatccttagc caagttactg agctactctg agcctaagtt 41760 ttgtcactgt agaatgaaac taataacagt gtctttctta caaagtagtt gtgaggataa 41820 aataattcat gtgaagtctt tattatactt tgtggcatct aatgagcact cagcatgtgt 41880 tatttacaag gaagagccag tatgattaga gaaacattgt agtagaagca tgcattaagc 41940 tttctggcaa tcctagagaa aggagcccag tctgtgttag aaaggaattg ttgtgaagag 42000 cttgacagaa aatgtaacgt ttgaggtgga ttttgaaaga ttagtcatgg ttgccaggca 42060 gagaaaagac aaagaggaaa tttgctttga ggtcaatgtc cacattcctt tatgtggtgg 42120 attataccat tcaattttac ttctaaattc cactgaaagg tggtattgtc atttcatttc 42180 aattaaataa attaaccttt tgtgtggaaa caactggtga ctgtgactaa acacatttaa 42240 ttctattgag tggagttgca gacttcttgt ggggcattaa cttagatttc tttccaattg 42300 cattaccacc acctcttttg taattatatg aaccagctgg ataggataat aaattgtttc 42360 ttaaaatgta ctggtgtctc tttactaaga actgctttgc gtatattgag tcacatacca 42420 aaggagaaaa gggtatattc tggagccgcg tgtagctgtc atcagattgt tatttcccag 42480 actttggatg cagtttttat gtttggatag agatttcaca aacaaagtga caaaactctg 42540 cagtttatct tcttattgtt atagatggct gatatggcaa agcatacctg gctagtttcc 42600 cagatcagtg cctttgttgc tcagagttta ataataaaca atttaaaatt ctagtagagt 42660 tattttactc agctggtaga ctgtcagtct tcttaggtag gctatggggt atcctgtgag 42720 tattcagttt cagaaaacat ttataaattt ataaatatag aaaaggacta gtagattaag 42780 ttgtttttgg gcaaacaaag tcatctactt tttaaaggaa ctcaaactat tacatatttt 42840 tacaggtgtt ttcaaaatat ttattttctt gaacatttat tttaagttcc agggtacacg 42900 tgcaggttgt acaggtttgt tacacaggta aacatgtgcc atggtagttt gctgcacaga 42960 tcaacccatc tccagcatcc attcgctatt cttcctgacg gtctccctcc cccaacccct 43020 cctaacaggc tgcattgtgc gttgttcccc ccaaccccat gtgtccatgt attctcatct 43080 ttcagctccc atacgtgaga acatgtggtg tttgggtttc cgttcctaca ttagtttgct 43140 gaggataatg gcttccagct ccatccatgt tcctgcaaag gacgtgatct catttctttt 43200 tatggctgca tagtattcca tggtgtatat ataccacatt ttctttatcc agtctgggaa 43260 tttgggttga ttccatgtct ttgctattgt gaatagtgct gcaatgaaca tacacatgcg 43320 tttgtcttta taatagaatg atttatattc ctttgggtat atacccagaa atggaattgc 43380 tgggtcaaat ggtatttcta cctctagatc tttgaggaat tgccacactg tctttcacaa 43440 tggttgaact aatttaccct cccaccaaca gtgtaaaagt gttctttttc tccacaactt 43500 tgccagcatc tgttgtttct ggacttttta ataatttcca ttctgactgg catgagatgg 43560 tatctcagtt gtggttttga tttgcatttc tctaatgatc agtgatgttg agcttttttt 43620 catatgtttg ttggcctcat gaatgtcttc ttttaagaag tgtctgttca tgtcctttgc 43680 ctgcctgctt tttaatgggg ttattttttc ttctaaattt gcctaagttc attgtagatg 43740 ctggatatta gacctttgtc agatggatag attacaaaca ttttctccca ttctgtgagt 43800 tgcctgttga ctctgatgct agtttatttt gctgtacaga cgctctttag tttaattaga 43860 tcccatttgt caatttatgc ttttgttgta attgcttttg aaatttttgt catgaaatct 43920 gcccatgcct atgtcctgaa tggtattgcc cagattttct tctagggttt tatagtttgg 43980 ggttttacat ttaagtcttt aattcacctt gagttaaatt ttgtattagg tgtaaggaag 44040 gggtccagtt tcagttttcc acatatggct agccagtttt cccagcacca tttattgagt 44100 agaaaatcct ttccccattg cttgtttttg tgaggtctgt cgaagatcag atagttgtag 44160 gcatgtggtc ttatttctga gttctcaatt ctgttccatt ggtctatgtg tctgttcttg 44220 taccagtacc acacgatttt ggttactgta gctttgtagt atagtttgaa gttgggtagc 44280 gaagtgcctt cagctttgtt ctttttgctt aggattgtct tggctatttg ggctctttat 44340 tggttccata tgaattttaa aatagtttat tctaattctg tgaagaatgt caatggtagc 44400 ttaatgggaa tagcattgaa tctgtaaatt actttgggca gtatggacat tttcacaata 44460 ctgattcttc ctatccatga gcatggaatg tttttcccat ttgtttgtgt cctctctgat 44520 ttccttgagc agtggtttgc agttctcctt gaagaggtcc ttaatttccc ttgttagctg 44580 tattcctagg tattttattc ttttgtagca attgtgaatg ggagttgatt tatgatttgg 44640 ctctctgctt gcatgttgtt ggtgtatagg aatgctagca atttttgcac attgattttg 44700 tatcctgaga ctttgctgaa gttgcttatc agcttaagaa gcttttggga tgaaacgatg 44760 ggattttcta gatatagggt catgtcatct gcaaacagag gtaatttgac ttctctccct 44820 atttgaatac cttttattta tttctcttgc ctgattgccc tggccagagc ttccagtact 44880 gtgttgaaca ggggtggtga aaaagggcat ccttgtcatt tgctggtttt caagggtaat 44940 gcttccagct tttcgccatt cagtatgata ttggctgtag gtttgtcaca tatggccctt 45000 actattttga ggcatgttcc ttcaatacct agtttattga gagtttttaa catgaaagga 45060 tgttgaattt tatcaaaggc tttttctgtg tctgttgagc tgatcatatg gttatgtctt 45120 tcgttctttt tatgttgatg aattacattt attgatctgc atatgttgaa ccaaccttgc 45180 atcctgggga tgaacctgac ttgattgtgg tggataagct ttttgatgtg ctgctggatt 45240 cagtttgcca gtgttttatt gaggattttt gcatcgatgt tcatcaggca tattggcctg 45300 aagttttctt tatgttgttg tacctctgct agattttggt attaggatta tgctggcctc 45360 ataaaataag ttagggagga gtcccttctt ttgaattttt tagaatagtt tcagtagcca 45420 tggtaccagc tcttctttat acctctggta aaattcagct ctaaatctgt ctggttgtgg 45480 gtttgttttt tggttggtga gctatttatt actgattcga tttcagaatt atgggtctat 45540 tcagggattt aatttcttct tgggtcagtc tttggagggt ttttgtatct aggaatttat 45600 ccatttcttc cagattttct actttatgtg tatagagctg tttatagtat ccttgcatgg 45660 tggtttgtat ttgtgtggga tcagtagtga taccccctta tcatttctga ttgtgtctga 45720 ttctcctctc tcttctcctt tattagtcta actagcagtc tatttttttt atttatttat 45780 ttatttattt atttatttat ttatttattt attttccaaa aaaccagctc ctggattcat 45840 tgattttttt ttgaaggatt tttcttgtct ctgtctcctt cagttctact ctgatcttgg 45900 ttatttcttg tcttctgcta gctgtgtttg ctcttggtac tctagttctt tttgttgcaa 45960 tatcaggttg ttaatttgag atctaagtag ctttttgtcg tgggcattta gtgctacaaa 46020 tttccctctt aacactgctt tagctgcgtc tcagagattc tgatatgttg tctcttcatt 46080 ctcattagtt tcaaagaact ccttgatttc tgccttaatt tcattattga cccaggagtc 46140 attcaggagc agattgttcg gtttccatat agttgtgtgg ttttgagtga atttcttagc 46200 cttgagttct aatttgattg cactgtgatc tgagagactg ttatgatttc agttctttgc 46260 atttgctgag aagtgtttta ccttactgtg atcagtttta gagtaaatgc catgtggcaa 46320 tgagaagaat gtatgttctg ttgttttggg gtgcagagtt ctgtagatat ctatcagttc 46380 tgtttgatcc agagctgagt tcaggtcctg aatatctttg ttaattttct gtcccaatga 46440 tctgtcaaat attgtcagtg gggcattaaa gtctcccaca attatcatgt gggagtctaa 46500 gtctctttat aggtctctaa gaacttgctt tatgaatctg cgtactcctg aattaggtgc 46560 atatatattt aggatagtta actcttgaat tgaacccttt accattatgc aatgcccttc 46620 tgtctttttt aattttggtt ggtttaaagt ctgttttgta agaaattagg attgtagccc 46680 ctgctttttt ctgttttcca tttgcttggt aaattttcct ccatcccttt atttcgagcc 46740 tatgtgtgtc tttgttcatg agatgggtcc cttgaagaca gcatactgat gggtctcaac 46800 tctttatccg gtttgccatt ctgtgtcttt taattgggtc atttagccta tttacattta 46860 aggttaatat tgttatgtat gaatttgatc ctgtcatgat gctagctggt tattttgcag 46920 acttgtttat gtggttgctt catagcatca ctggtctgtg tactttagtg tgtttttgta 46980 gtggctggta atggttgttc ctttccatat ttaatgcttt cttcaggagc tcttgcaagg 47040 caggcctggt ggtgacgaat tccctcagta tttgtttgtc tgaaaaagat cttatttctt 47100 cttcacttat gaagcttggt ttggccagac atgaaattct gggttggaat ttcttttctt 47160 taagaatgtt gaaaattggc ccccaatctc ttctggctgg tagggtttct actgagaggt 47220 ccactgttag tctgactggt ttctcttcgt aggtgacctg gcctttctct ctggctgtcc 47280 ttaacatttt ttcttttatt ttgaccttgg agaatctgaa gattatgtgt cttgaagttg 47340 cttttctcat ggagtacctt actggggttc tccggatttc ctgaatttga gttttggcct 47400 gtcttgctag gttggggaag ttctcctgga tgataatctt gaagtatgtt ttccaacttg 47460 gttctgtttt ccttgtcttt ttcaggtacc ccaattagtc ataggttttt acatagtccc 47520 atatttcttg gaggttttgt tcattccttt tcattctgtg tgtgtgtgtg tgtgtttgtg 47580 tgtgtgtgtg tattattgtc tgcctgtctt atttgagaaa gatacttggt ctattctgct 47640 attaatactt gtgattgcat tatgtatttc tcatgttgta tttttcagct ccatcaggtc 47700 agtaatgctc ctctctaaac tggctattct ggctgttggc tcctgtattg ttttatcatg 47760 attcttaact tatttgcatt gggttacagc atgctccttt agctcagtga agttcattat 47820 tactcacctt ctgaagccta cttctgtcag ttcagccatc tcaacttcag cccagttctg 47880 tgcccttgct ggagaggtgt tgctgtcatt tggaggagaa gcagcactct ggcttttaga 47940 gttttccagg tttttgcatt gattctttct catctttgtg ggcttatctg tcttagattt 48000 ttgaggtggc ttacctttca gtggggtttt gtggggtctt ttttgttgat gttgttgttg 48060 ttactttgtt tgtttgttgt ttttctttta acagtcagac cacttttccg taggactgtt 48120 gcagtttgct gggggattgc tccagaccct agttgtttca gtttttacct tacctggagg 48180 tatcaccagt taaggctgca aaacagcaat gatggtagcc tgttccttcc tttgggagct 48240 ctgtcctaga ggggtactga cctgttgcca gcccagacac tcctgtagga gttgtctaga 48300 gacccctgtt gggaggtctc acccagtcag gagagacagg atcagggacc tgcttaaagc 48360 agtctggctg ctttttggta gagtaggtgt gctgcactgt ggggatccca tccccatccg 48420 gactgcttca actgtccaga gccaacagtc tggaaaggct gtgtcgactg aactgcttag 48480 atgttggcca cccctcccgc caggggctcc atccctggga gggatcagag atctgtctgt 48540 ataaccctgg ctggagttcc tgaaattcct ggagttgctg gtgctcactg gcaccaactg 48600 gtgaggaggg atggatcggg gtcccactta aagaaaaagt ctggccacaa tctggcacag 48660 cagctatgct gcattatggt agactcctcc ttgtctagac tgcctggact ccctggagct 48720 ggcaggctgg aatggctgtg tcaaatgaac tgcagaaaca ggggccgtct ctccccaggg 48780 ctctatccca gggagagatc agagtcctgt tcatataacc ctggctagag ttgttgaaat 48840 tcctgcaggg aggcaccacc cagtgaggag ggatggatta gggtcccact taaagaagca 48900 gtgtggctgt gatctggcac agcagctgtg ttgcattgtg ggcgactcct ccttgtccag 48960 tggactcctt ggagctggca gactagaatg gctgagtcga cctaaccaca gaaatggcag 49020 ctgcccctcc ccccaggaac ttgtccatct caggcagtct ccagcctgtt gcactggttg 49080 gctggatttc caatccagtg aggcttaact tgtgagatgc catggaagtg gggcccgcag 49140 aacaacaccg cttggctccc tggattcagc tcccttcctt ggagaattta tggatggatc 49200 tcctgcctta ccgggattcc tggggctaga gtctattaaa ctcctgggtc tctgtgtgag 49260 cctgagcaac tgctctgcca agactctgca cagctctgtg tattggaccc aaggccctgg 49320 tggtgtgggc tcacgagggt atctcctgat ccacaggctg caaagatccg tgagagaaac 49380 gtggtttccc aggtgggggt cacacaatca ctcactgctt cccgtggctg ggagtggggg 49440 tttctttggc tccatgccac tcccaggtgg gcagtcactc catcctgatt tccttagttc 49500 tttctccatg ggtcgagttg tccgcctagt cagtcccaat gtgaaaacct ggatatttca 49560 gttgaaggtg ctgaattcac ttgccatttt cattcctctg ggagaactgc agaccacagc 49620 tgcttctaat cagtcatctt tgccaaaata tctgtttagt ttgttaccaa cttccttcag 49680 aaaattccat atggaaaccc aacacctaca aatacacttt tgaaaagata acaggacaaa 49740 ttgtttgcat tttataattt cctatgttta attttgtcta ttgttataat ttaatacttc 49800 tgaaaaattt ttttaattga ctaatgaaag cagctctcag gtgtggttaa tcttataatt 49860 gcataaataa ggaaaaccag ttatgtttgt aaataaatat taaaggactg atgtttatac 49920 acttattttg tatatggatc ataatgaaaa ttaaatttag catatgataa tataaatagg 49980 aaaaatcact tactcaaaat ttgcatttgt actataaatc catgtcttcg tatgattttt 50040 aaactcttaa agattatagt aatcttttca ataaggactt tataaaagtt taaaaatgga 50100 acagagaaat tgaattcaaa ttattaagtt actaaataca ttcttatata ggcacaaggt 50160 ggcttatatt tatactttca aaaagacttt ggagttgtca cttaactgtt ctactttata 50220 aagatttgat gttaatgttt ttttttcacc aaaaatttag atatagtgtt tgtagctatt 50280 tcttggtgaa cagttatttt tattttaaat tggaaaaata atcctcatca gtcaggagaa 50340 aacaaaataa tgattcatga agaaatatgt tgattttttc tttcagacct gaagattatc 50400 ggctataaga gaataagaat tgcagaaaat aacagtgaag tgattgaaac tttcttctga 50460 tgagtttctc taacctacag gatggagtaa aacaactgct acagttcagc acctgtttta 50520 tgtgccgaat cactgtgggg aaaggtcagg aaggtatagt ccttcaatag gaaattgtaa 50580 ttaaaatata attttataga accattttta tgtaatctga tttgaatgtt atagttgata 50640 ataataaaat cacttacttg gttgactatt tagtgttgca tttaatgata aaaaacagac 50700 cctgtgtata gaattgcagt tacacattcc aaaaatgcct ctccatgcag attttctcaa 50760 gtttgcaggt cactattctt gtcccttgtg aacaagctct accttcactt ttcatttcag 50820 tggctttttt tatctaactt gtcatccatc taaattcata tttgcagctt agtagggtaa 50880 agtttgccct gaaatattta taaactgcta tctttttctg tgtcccatat agcctcttgg 50940 ccccacaatg acaccagaat acataatgcc tgtctgctcg ttagaaatcc ctcttccctg 51000 ccagtgattc tgtttgtaga ctctccacac tgttttccca tatctgtcct gttggcttcc 51060 attcatctct gtactggcac ttagattggg gtgctgggat gactattgct tgtaaaatat 51120 gctctctaga aattaagata ctgagccact gcatttgagc attttcatgg tggtattttt 51180 tcatctcttg ccatagccaa tcaaggaatg aatttcagac caactttccc attcattcag 51240 caaacatata ttcagtactg ctatatacca ggcacagtgc taggtcctgg gaaaacagaa 51300 atcatacaag ttgcccccaa cacttccgtg ccccatcaaa gagtctattg taggatggga 51360 aggaagtata gatagatctg taaacaagta aaactggttg tcaaatgtgc taatacaact 51420 gtgtgaatta tgatgtaaac atgtttattt aataactgca ttaaatatga cataagcacc 51480 tactatgtgc caggcacaac cactttatat gaattattta attaatacat actgtaatgg 51540 aggtactgtt atcctcccaa tttaaagggg acacagtgaa gcagcaataa attaagtaat 51600 ttggctaagg taaaatcagg atttgcatcc aggcagttgg acttgaaagc ccatggactt 51660 acccactgct agatgagaaa aggtgaatat gtttctgtat tacaaattct ttaacaaagc 51720 acgtataacc cggcacaggt taacaactac cattcaaaag gaagtctcag ttacatttta 51780 aggtgttgga ccaaaaagaa cctgcccgtc ttacttttga tggagtaatt ttaggggagt 51840 gagccaatat gaaataagtc tccagttcct gatacatcag ctttaatagg aatttttgcc 51900 cagtgtatac caagcaccta gaacagtgct ggcacatgtt tgttggatgg tggaaggacc 51960 actgaagaaa ggtccttatt tttaacaaaa cttgagtgtt t 52001 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 12 agaaactcga catattcctc 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 13 gagaaactcg acatattcct 20 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 14 ctagcaaaag tggcccattg 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 15 catattctag caaaagtggc 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 16 tttcccatct aagagatacc 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 17 ttttcccatc taagagatac 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 18 ggtggctgca ttttcccatc 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 19 aggtggctgc attttcccat 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 20 tgccaggtgg ctgcattttc 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 agtttgccag gtggctgcat 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 agcaagtttg ccaggtggct 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 tgccatagca gcaagtttgc 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 atgccatagc agcaagtttg 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 agtcttatag atgccatagc 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 ggtttatgta atccctgaag 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 aggtttatgt aatccctgaa 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 tacacaggtt tatgtaatcc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 ggtacacagg tttatgtaat 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 atggtacaca ggtttatgta 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 tgcatggtac acaggtttat 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 agtgcatggt acacaggttt 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 actcagtgca tggtacacag 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 ccacagtcac tcagtgcatg 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 taacaacatg atccccacag 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 tgtgttcatt ataacaacat 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 gtcttgtgtt cattataaca 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 atgcaatgtg tcttgtgttc 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 aaaatgcaat gtgtcttgtg 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 gcgaagagta tactttttgt 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 gtatgcgaag agtatacttt 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 agtatgcgaa gagtatactt 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 cagtatgcga agagtatact 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 gggtagccag tatgcgaaga 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 acctgggtag ccagtatgcg 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 tgagcagctg ttacttgtct 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 ctgagcagct gttacttgtc 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 cactggatcc ctcaggtgaa 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 ttgccactgg atccctcagg 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 gttttacaat tgccactgga 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 gccataaata gctagtttta 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 tggcagcatg ccataaatag 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 tttttggcag catgccataa 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 tcattgttct tctgtgaagg 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 ccatcattgt tcttctgtga 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 tgcaaccttt ccatcattgt 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 gaatatcttc tggaatatac 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 actaaattct taagaatatc 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 ctctactaaa ttcttaagaa 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 tcgtggttga ggaagctcct 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 aggtattttt cgtggttgag 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 ttgtgtactc atctagacgt 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 ttcttgtgta ctcatctaga 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 ggagtccaca atcttggaaa 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 gtggagtcca caatcttgga 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 tcaggtggag tccacaatct 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 tcttcaggtg gagtccacaa 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 gatattcttc aggtggagtc 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 tatagccgat attcttcagg 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 tctcttatag ccgatattct 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 ttcaatcact tcactgttat 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 tcatcagaag aaagtttcaa 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 acgttaaaga aactcatcag 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 gctgaactgt accatttgtt 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 gtgctgaact gtaccatttg 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 caggtgctga actgtaccat 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 aacaggtgct gaactgtacc 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 cacataaaac aggtgctgaa 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 cctttcccca cagtgattca 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 aggactacac cttcctgacc 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 gaaggactac accttcctga 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 gattacataa aaatggttct 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 ttacccagta tgcgaagagt 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 attatcttac ccagtatgcg 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 85 aacttactgc cactggatcc 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 86 agaaacttac tgccactgga 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 87 cctcatctgg aaaaagatgc 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 88 aagaggatgc tcaccaatgc 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 89 tgggcagatt tcatgacaaa 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 90 gaggaatatg tcgagtttct 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 91 aggaatatgt cgagtttctc 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 92 caatgggcca cttttgctag 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 93 gccacttttg ctagaatatg 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 94 ggtatctctt agatgggaaa 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 95 gtatctctta gatgggaaaa 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 96 gatgggaaaa tgcagccacc 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 97 atgggaaaat gcagccacct 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 98 gaaaatgcag ccacctggca 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 99 atgcagccac ctggcaaact 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 100 agccacctgg caaacttgct 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 101 gcaaacttgc tgctatggca 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 102 caaacttgct gctatggcat 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 103 gctatggcat ctataagact 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 104 cttcagggat tacataaacc 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 105 ttcagggatt acataaacct 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 106 ggattacata aacctgtgta 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 107 attacataaa cctgtgtacc 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 108 tacataaacc tgtgtaccat 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 109 ataaacctgt gtaccatgca 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 110 aaacctgtgt accatgcact 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 111 ctgtgtacca tgcactgagt 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 112 catgcactga gtgactgtgg 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 113 ctgtggggat catgttgtta 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 114 atgttgttat aatgaacaca 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 115 tgttataatg aacacaagac 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 116 gaacacaaga cacattgcat 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 117 cacaagacac attgcatttt 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 118 acaaaaagta tactcttcgc 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 119 aaagtatact cttcgcatac 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 120 aagtatactc ttcgcatact 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 121 agtatactct tcgcatactg 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 122 tcttcgcata ctggctaccc 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 123 cgcatactgg ctacccaggt 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 124 agacaagtaa cagctgctca 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 125 ttcacctgag ggatccagtg 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 126 cctgagggat ccagtggcaa 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 127 tccagtggca attgtaaaac 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 128 taaaactagc tatttatggc 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 129 ctatttatgg catgctgcca 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 130 ttatggcatg ctgccaaaaa 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 131 ccttcacaga agaacaatga 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 132 tcacagaaga acaatgatgg 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 133 acaatgatgg aaaggttgca 20 <210> SEQ ID NO 134 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 134 gtatattcca gaagatattc 20 <210> SEQ ID NO 135 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 135 aggagcttcc tcaaccacga 20 <210> SEQ ID NO 136 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 136 ctcaaccacg aaaaatacct 20 <210> SEQ ID NO 137 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 137 acgtctagat gagtacacaa 20 <210> SEQ ID NO 138 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 138 tctagatgag tacacaagaa 20 <210> SEQ ID NO 139 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 139 tttccaagat tgtggactcc 20 <210> SEQ ID NO 140 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 140 tccaagattg tggactccac 20 <210> SEQ ID NO 141 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 141 agattgtgga ctccacctga 20 <210> SEQ ID NO 142 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 142 ttgtggactc cacctgaaga 20 <210> SEQ ID NO 143 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 143 cctgaagaat atcggctata 20 <210> SEQ ID NO 144 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 144 agaatatcgg ctataagaga 20 <210> SEQ ID NO 145 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 145 ataacagtga agtgattgaa 20 <210> SEQ ID NO 146 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 146 ttgaaacttt cttctgatga 20 <210> SEQ ID NO 147 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 147 atggtacagt tcagcacctg 20 <210> SEQ ID NO 148 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 148 ggtacagttc agcacctgtt 20 <210> SEQ ID NO 149 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 149 ttcagcacct gttttatgtg 20 <210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 150 tgaatcactg tggggaaagg 20 <210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 151 ggtcaggaag gtgtagtcct 20 <210> SEQ ID NO 152 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 152 tcaggaaggt gtagtccttc 20 <210> SEQ ID NO 153 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 153 agaaccattt ttatgtaatc 20 <210> SEQ ID NO 154 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 154 actcttcgca tactgggtaa 20 <210> SEQ ID NO 155 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 155 cgcatactgg gtaagataat 20 <210> SEQ ID NO 156 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 156 ggatccagtg gcagtaagtt 20 <210> SEQ ID NO 157 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 157 tccagtggca gtaagtttct 20 <210> SEQ ID NO 158 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 158 gcattggtga gcatcctctt 20 <210> SEQ ID NO 159 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 159 tttgtcatga aatctgccca 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding mitochondrial ribosomal protein L13, wherein said compound specifically hybridizes with said nucleic acid molecule encoding mitochondrial ribosomal protein L13 (SEQ ID NO: 4) and inhibits the expression of mitochondrial ribosomal protein L13.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding mitochondrial ribosomal protein L13 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of mitochondrial ribosomal protein L13.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding mitochondrial ribosomal protein L13 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of mitochondrial ribosomal protein L13.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding mitochondrial ribosomal protein L13 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of mitochondrial ribosomal protein L13.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding mitochondrial ribosomal protein L13 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of mitochondrial ribosomal protein L13.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of mitochondrial ribosomal protein L13 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of mitochondrial ribosomal protein L13 is inhibited.
 19. A method of screening for a modulator of mitochondrial ribosomal protein L13, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding mitochondrial ribosomal protein L13 with one or more candidate modulators of mitochondrial ribosomal protein L13, and b. identifying one or more modulators of mitochondrial ribosomal protein L13 expression which modulate the expression of mitochondrial ribosomal protein L13.
 20. The method of claim 19 wherein the modulator of mitochondrial ribosomal protein L13 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of mitochondrial ribosomal protein L13 in a sample using at least one of the primers comprising SEQ ID NOs 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with mitochondrial ribosomal protein L13 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of mitochondrial ribosomal protein L13 is inhibited.
 24. The method of claim 23 wherein the disease or condition involves a defect in a mitochondrial process. 